大黄素和维生素C对团头鲂生长、非特异性免疫以及抗应激的影响
|
摘要
大黄素(Emodin)是从中草药大黄(Rheum officinale Baill)根茎中提取的主要有效成分,具有抗菌消炎、抗病毒、抗氧化以及清除氧自由基、降血脂、保护肝脏和免疫调节等多种功效。维生素C(Vc)是鱼类必需的营养素,兼有免疫增强和抗氧化的双重功效,但大多数鱼类缺乏合成Vc所需的L-古洛糖酸内酯氧化酶(L-gulonolactone oxidase, GLO),必须从食物中摄取Vc。为了研究大黄素和高剂量Vc对鱼类生长、非特异性免疫以及抗应激的影响,选用我国主要淡水养殖鱼类之一的团头鲂(Megalobrama amblycephala, Yih)作为研究对象,首先分离、克隆出团头鲂两种热休克蛋白70 cDNA的全长序列,并对其基因结构和表达特征进行了分析。然后,在此基础上构建了团头鲂两种HSP70s的原核表达系统,并优化了表达条件,对表达产物进行了纯化和鉴定。同时,选取1200尾健康的团头鲂,体质量(133.44±2.11)g,随机分成4组,其中1组为对照组,投喂基础日粮(Vc含量50.3mg/kg),另外3组为试验组,投喂饲料是在基础日粮中分别添加60 mg/kg大黄素、700 mg/kg Vc、60 mg/kg大黄素+700 mg/kg Vc,每组设3个重复。连续投喂60 d后结束饲养试验,取鱼进行拥挤胁迫、高温应激和感染嗜水气单胞菌的应激试验。
1团头鲂两种热休克蛋白70基因克隆与序列分析
采用RT-PCR和cDNA末端快速扩增法(rapid amplification of cDNA ends, RACE),分别从室温和34℃热激团头鲂的肝脏中克隆到两种热休克蛋白70基因,分别命名为Ma-HSC70和Ma-HSP70基因。它们的cDNA全长分别为2336和2224 bp[不包括poly(A)],分别含有1950和1932 bp的开放阅读框,编码649和643个氨基酸,分子量约为71.24和70.52 kD,理论等电点为5.25和5.30。Ma-HSC70基因在编码区含有7段内含子,所有内含子均遵守GT/AG法则,为组成型HSC70,而Ma-HSP70基因在编码区不含内含子,为诱导型HSP70。氨基酸序列分析表明,Ma-HSC70和Ma-HSP70均含有HSP70家族的3个签名序列,两个部分重叠的双向核定位信号序列(nuclear localization signal sequence, NLS)以及胞质的特征基序EEVD等。同源性分析表明,Ma-HSC70与异育银鲫等脊椎动物HSC70的氨基酸序列相似性高达93.0%以上,而Ma-HSP70与鲫鱼等脊椎动物HSP70氨基酸序列的相似性高达85.0%以上,它们之间氨基酸序列相似性为86.5%。生物信息学分析显示,团头鲂两种HSP70s基因所编码的蛋白以亲水性区域为主,均具有丰富的B细胞抗原位点,无信号肽,为非典型的分泌型蛋白,无明显的跨膜区域；同时还含有众多的蛋白激酶C磷酸化位点、N-肉豆蔻酰化位点、酪蛋白激酶Ⅱ磷酸化位点和N-糖基化位点,推测这些位点在蛋白质折叠,转运,细胞内定位以及细胞信号转导与调控中发挥重要作用。这两种蛋白的二级结构均以a-螺旋和无规卷曲为主,空间结构包括N端ATPase功能域和C端多肽结合功能域。
2大黄素和维生素C对团头鲂生长、生理生化指标以及两种HSP70s mRNA表达的影响
饲养试验结束后,测定团头鲂的生长性能,并采样检测鱼的肌肉成分、血液和肝脏生化指标以及肝脏两种HSP70s mRNA的表达水平。结果表明,与对照组相比,大黄素、Vc组显著提高了鱼体增重率和特定生长率,血清中总蛋白(TP)、溶菌酶(LSZ)和碱性磷酸酶(AKP)的水平,肝脏中超氧化物歧化酶(SOD)的活性和诱导型HSP70 mRNA的基础表达水平,降低了饵料系数、死亡率、血清中皮质醇(COR)、甘油三酯(TG)以及肝脏丙二醛(MDA)的含量(P<0.05)；配伍组虽然血清中TP、LSZ的含量以及肝脏HSP70 mRNA的水平显著升高,肝脏MDA的含量也显著降低(P<0.05),但均未表现出协同增效作用。此外,与对照组相比,大黄素组还显著提高了团头鲂肝脏过氧化氢酶(CAT)的活性(P<0.05),而Vc和配伍组CAT的活性与对照组的差异不显著(P>0.05)；其它一些指标如血清中葡萄糖(GLu)、谷丙转氨酶(GPT)、谷草转氨酶(GOT)、胆固醇(CHOL)、肝脏组成型HSC70 mRNA的表达水平、鱼体形体指标以及肌肉组成成分等各组之间差异均不显著(P>0.05)。
3大黄素和维生素C对团头鲂在拥挤胁迫下的生化指标及两种HSP70s mRNA表达的影响
饲养试验结束后,从各池中取25尾规格基本一致的团头鲂,进行连续48 h的拥挤胁迫(100g/L)试验,分别于0h、12h、24h、48h取样分析团头鲂血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平,并统计各组鱼的累积死亡率。结果表明,在拥挤胁迫后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清中TP和AKP的水平,肝脏中SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清中COR、GLU、GPT、GOT、TG以及肝脏MDA的水平,而LSZ的活性表现为先下降后升高；配伍组中,这些指标虽然有类似以上的变化趋势,但大多差异不显著(P>0.05),且同样未表现出协同增效作用。此外,各组鱼血清CHOL的水平在拥挤胁迫前后的差异均不显著(P>0.05)。统计表明,大黄素和Vc组鱼的累积死亡率在拥挤胁迫24h、48h均显著低于对照组(P<0.05),而配伍组与对照组的差异均不显著(P>0.05)。
4大黄素和维生素C对团头鲂在高温应激下的生化指标及两种HSP70s mRNA表达的影响
饲养试验结束后,从各池中取25尾规格基本一致的鱼进行高温应激(34℃)试验。取样检测团头鲂在高温应激前后血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平,并统计各组鱼在高温应激下的累积死亡率。结果表明,在高温应激后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清TP、LSZ和AKP的水平,肝脏SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清COR、GLU、GPT、GOT、TG以及肝脏MDA的水平；配伍组中,这些指标虽然也有类似以上的变化趋势,但大多差异不显著(P>0.05),且未表现出协同增效作用。此外,各组鱼血清胆固醇的含量在高温应激前后的差异均不显著(P>0.05)。统计表明,在高温应激下,Vc组鱼的累积死亡率均显著低于对照组,大黄素组鱼的累积死亡率除6h外,也显著低于对照组(P<0.05),而配伍组与对照组的差异均不显著(P>0.05)。
5大黄素和维生素C对团头鲂感染嗜水气单胞菌后的生化指标及两种HSP70s mRNA表达的影响
饲养试验结束后,从各池中取15尾团头鲂进行腹腔注射感染嗜水气单胞菌试验,并在攻毒前后采样检测团头鲂血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平；另从各池中取10尾鱼进行同样的攻毒试验,并统计攻毒后鱼的累积死亡率。结果表明,经攻毒后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清TP.LSZ和AKP的水平,肝脏SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清COR、GLU、GPT、GOT、TG以及肝脏MDA的水平；配伍组中,这些指标虽然有类似上述的变化趋势,但大多与对照组的差异不显著(P>0.05),且未表现出协同增效作用。此外,各组鱼血清胆固醇的含量在攻毒前后的差异均不显著(P>0.05)。统计也表明,经攻毒后,大黄素和Vc组鱼的累积死亡率在24 h、48 h均显著低于对照组(P<0.05),而配伍组鱼的累积死亡率与对照组的差异均不显著(P>0.05)。
6团头鲂两种热休克蛋白70的原核表达、纯化及鉴定
采用PCR方法扩增团头鲂组成型HSC70和诱导型HSP70基因完整的编码区片段,并分别克隆到原核表达载体pET-22b(+)中,经双酶切和DNA测序鉴定后,将这两种重组表达质粒分别转化大肠杆菌BL21 (DE3),用1 mmol/L IPTG在不同温度及时间下进行诱导表达,并对表达产物进行SDS-PAGE分析,建立最佳诱导表达条件。采用Ni-NTA His Bind Resins亲和层析和DEAE-Sepharose FF阴离子交换柱层析对目的蛋白进行纯化,并进行Western-blotting验证。结果表明,成功构建了团头鲂两种重组表达质粒pET-22b(+)/Ma-HSC70和pET-22b(+)/Ma-HSP70,表达融合蛋白的相对分子量均约为72 kD,并能与兔抗人HSP70多抗进行特异性结合,这两种HSP70s融合蛋白经纯化后的纯度均达到95%以上。在较高温度(37℃)下,有利于融合蛋白的快速大量表达,但易形成包涵体；在较低温度(25℃)下,有利于融合蛋白的可溶性表达,但也会降低融合蛋白的表达速度。综合各种因素,本实验选择融合蛋白Ma-HSC70在25℃和Ma-HSP70在30℃下分别诱导7h作为可溶性表达的最佳条件。
总之,本研究首次从团头鲂肝脏中克隆到两种HSP70s基因,并对该基因的结构和表达特征进行了分析,确定Ma-HSC70基因为组成型,而Ma-HSP70基因为诱导型。在此基础上成功构建了团头鲂两种HSP70s的原核表达系统,并优化了表达条件,最后得到了纯度在95%以上的两种热休克蛋白70的融合蛋白。这些为进一步研究这两种蛋白的结构和功能以及相应抗体的制备打下了基础。饲养和应激试验则表明,在基础日粮中添加大黄素60mg/kg或Vc 700 mg/kg可促进团头鲂的生长,降低饵料系数,提高鱼体的非特异性免疫力、抗氧化和抗应激能力,减少应激死亡率。二者配伍则效果不佳,其相互作用的机理还有待于进一步的研究。
Emodin is an active anthraquinone constituent extracted from the rhizome of rhubarb Rheum officinale Baill. It has many functions such as anti-bacterial and anti-inflammatory, antivirus, antioxidation and scavenging free radicals, reducing blood lipid, protecting the liver, and regulating immunity. Vitamin C (Vc), also known as L-ascorbic acid, is an essential nutrient for normal physiological functions in fish. Vc is an important immunostimulant and antioxidant. but most teleosts are unable to synthesize ascorbic acid due to lack of L-gulonolactone oxidase (EC 1.1.3.8) responsible for the de novo synthesis of Vc. Therefore, exogenous Vc is required in fish diet. In order to study the effects of dietary emodin, high-dose Vc and their combination on growth, non-specific immunity and anti-stress of fish, Wuchang bream (Megalobrama amblycephala, Yih), one of the major freshwater aquaculture fish, was selected as the research subject. Firstly, two full-length cDNA sequences of two HSP70s genes were isolated and cloned from the liver of Wuchang bream, and their gene structure and expression characteristics were analyzed. Then, two HSP70s prokaryotic expression systems were constructed on the basis of above two HSP70s genes, and the expression conditions were optimized. The expressed products were purified and identified. At the same time,1200 healthy fish with initial body weight of (133.44±2.11) g were selected and randomly divided into four groups:a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin,700 mg/kg Vc, and the combination of 60 mg/kg emodin+700 mg/kg Vc, respectively. Each diet was tested in triplicate. After continuous feeding for 60 days, the rearing experiment was over. Then, the experiments of crowding stress, heat stress and infected with Aeromonas hydrophila were carried out.
1 Molecular cloning and sequence analysis of two HSP70 genes in Wuchang bream
Two full-length cDNA sequences of two HSP70s genes were isolated from the liver of Wuchang bream using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70 genes, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly(A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kD, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule, belonging to the constitutive HSC70 whereas Ma-HSP70 gene did not contain any intron in the coding region, belonging to the inducible HSP70. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinaseⅡphosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins wereα-helix and random coil, and their spatial structures included N-terminal ATPase domain and C-terminal polypeptide-binding domain.
2 Effects of emodin and vitamin C on growth, physiological and biochemical parameters, and two HSP70s mRNA expression of Wuchang bream
At the end of the rearing experiment, the growth performance, muscle composition, biochemical parameters of serum and liver, and the mRNA levels of liver two HSP70s genes were investigated. The results showed that compared with the control, the weight gain and specific growth rate, the levels of total protein (TP), lysozyme (LSZ) and alkaline phosphatase (AKP) in serum, the activity of superoxide dismutase (SOD) and basal expression level of inducible HSP70 mRNA in liver significantly increased (P<0.05) while feed coefficient, mortality, the contents of serum cortisol (COR) and triglycerides (TG), and liver malondialdehyde (MDA) decreased (P<0.05) in emodin and Vc groups. Although serum TP, LSZ levels, and the mRNA level of liver HSP70 gene increased significantly (P <0.05), and liver MDA content also significantly decreased (P<0.05) in combination group, the synergism didn't appear. In addition, liver catalase (CAT) activity was also significantly improved in emodin group compared with the control (P<0.05) while the CAT activities in Vc and combination groups were not significantly different with the control (P> 0.05). The other indices, such as serum glucose (GLU), glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), cholesterol (CHOL), the mRNA level of liver constitutive HSC70, the fish body indices, and muscle composition were not significantly different among all groups (P> 0.05).
3 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream under crowding stress
At the end of the rearing experiment,25 fish per tank were exposed to crowding stress (100 g/L) for 48 h, the biochemical parameters of blood and liver, mRNA levels of liver two HSP70s genes, and cumulative mortalities of fish in each group were investigated at 0 h,12 h,24 h and 48 h, respectively. The results showed that, after crowding stress, compared with the control, serum TP and AKP levels, liver SOD and CAT activities, and the expression levels of liver HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees, and serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent, while the LSZ activity decreased at first and then increased significantly (P<0.05). In combination group, although these parameters had similar changing trend as above ones, most of them were not significant differences with the control(P>0.05), and did not show any synergism either. In addition, serum CHOL levels among all groups before and after crowding stress were not significantly different (P> 0.05). The statistics showed that, the cumulative mortalities of the emodin and Vc groups were significantly lower than that of the control at 24 h and 48 h (P<0.05), but there was no significant difference between the combination and control groups (P> 0.05).
4 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream under high temperature stress
At the end of the rearing experiment,25 fish per tank were exposed to high temperature stress of 34℃. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that, after heat stress, compared with the control, serum TP, LSZ, and AKP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. In addition, serum cholesterol levels among all groups before and after heat stress were not significantly different (P>0.05). Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P<0.05) while the difference between the combination and control groups was not significant (P>0.05).
5 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream infected with Aeromonas hydrophila
At the end of the rearing experiment,15 fish per tank were intraperitoneally infected with A. hydrophila (BSK-10), and the biochemical parameters of serum and liver, the mRNA expression levels of liver two HSP70s genes before and after infection were determined. Another ten fish taken from each tank were challenged with A. hydrophila as the same method as above, the cumulative mortality of each group was counted at different time point of post-infection. The results showed that after infection of fish, compared with the control, serum TP, LSZ and AKP levels, liver SOD, CAT activities, and the expression levels of HSC70 and HSP70 mRNA increased in emodin and Vc groups in varying degrees, while serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent. In combination group, although these parameters had similar changing trend as above ones, most of them were not significant differences with the control(P>0.05), and did not show any synergism either. In addition, serum cholesterol levels among all groups before and after infection were not significantly different (P>0.05). Statistics also showed that after infected with A. hydrophila, the cumulative mortalities of emodin and Vc groups were significantly lower than that of the control in 24 h and 48 h (P<0.05), but the difference of the cumulative mortality between the combination and control groups was not significant (P>0.05).
6 Prokaryotic expression, purification and identification of two HSP70s in Wuchang bream
The fragments of constitutive HSC70 and inducible HSP70 ORFs of Wuchang bream were amplified by PCR, and then cloned into prokaryotic expression vectors of pET-22b(+), respectively. The two recombinant plasmids, determined by double-endonuclease digestion and sequencing, were transformed into competent E. coli BL21 (DE3), respectively. The two recombinant strains were induced by 1 mmol/L IPTG at different temperatures and times, establishing optimal conditions for inducible expression. The expression products were purified by Ni-NTA His Bind Resins affinity chromatography and DEAE-Sepharose FF anion-exchange chromatography, and detected by SDS-PAGE and Western-blotting, respectively. The results show that two recombinant expression plasmids of pET-22b(+)/Ma-HSC70 and pET-22b(+)/Ma-HSP70, each expressing a fusion protein of about 72 kD identified by rabbit source anti-HSP70 polyclonal antibody, were successfully constructed, and the purity of two purified fusion proteins was more than 95%. A higher temperature (37℃) is conducive to the rapid expression of fusion protein, but it is easy to form inclusion body. A lower temperature (25℃) is in favor of the soluble expression of fusion protein, but it could also reduce the expression rate of fusion protein. All things considered, the optimal conditions for soluble expression of the two fusion proteins of Ma-HSC70 and Ma-HSP70 are induced for 7 h at 25℃and 30℃, respectively.
In conclusion, two full-length HSP70 cDNAs were cloned and characterized from Wuchang bream liver for the first time. There are seven introns in the coding region of Ma-HSC70 gene while there is no intron in the Ma-HSP70 gene, suggesting constitutive and inducible expression, respectively. Through optimizing the expression conditions, two purified fusion proteins of Ma-HSC70 and Ma-HSP70, which purities were more than 95%, have been obtained by prokaryotic expression systems of two HSP70s genes of Wuchang bream. Thus it laid a foundation for further study on molecular structure and function of the two proteins and associated antibodies. The rearing and stress experiments indicate that, the basal diet supplemented with either 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, lower FCR, increase non-specific immunity, antioxidant capacity, and stress resistance, reduce stress mortality. However, the combination of emodin and high-dose Vc showed no better effects. The mechanism of their interaction needs to be further studied.
1团头鲂两种热休克蛋白70基因克隆与序列分析
采用RT-PCR和cDNA末端快速扩增法(rapid amplification of cDNA ends, RACE),分别从室温和34℃热激团头鲂的肝脏中克隆到两种热休克蛋白70基因,分别命名为Ma-HSC70和Ma-HSP70基因。它们的cDNA全长分别为2336和2224 bp[不包括poly(A)],分别含有1950和1932 bp的开放阅读框,编码649和643个氨基酸,分子量约为71.24和70.52 kD,理论等电点为5.25和5.30。Ma-HSC70基因在编码区含有7段内含子,所有内含子均遵守GT/AG法则,为组成型HSC70,而Ma-HSP70基因在编码区不含内含子,为诱导型HSP70。氨基酸序列分析表明,Ma-HSC70和Ma-HSP70均含有HSP70家族的3个签名序列,两个部分重叠的双向核定位信号序列(nuclear localization signal sequence, NLS)以及胞质的特征基序EEVD等。同源性分析表明,Ma-HSC70与异育银鲫等脊椎动物HSC70的氨基酸序列相似性高达93.0%以上,而Ma-HSP70与鲫鱼等脊椎动物HSP70氨基酸序列的相似性高达85.0%以上,它们之间氨基酸序列相似性为86.5%。生物信息学分析显示,团头鲂两种HSP70s基因所编码的蛋白以亲水性区域为主,均具有丰富的B细胞抗原位点,无信号肽,为非典型的分泌型蛋白,无明显的跨膜区域；同时还含有众多的蛋白激酶C磷酸化位点、N-肉豆蔻酰化位点、酪蛋白激酶Ⅱ磷酸化位点和N-糖基化位点,推测这些位点在蛋白质折叠,转运,细胞内定位以及细胞信号转导与调控中发挥重要作用。这两种蛋白的二级结构均以a-螺旋和无规卷曲为主,空间结构包括N端ATPase功能域和C端多肽结合功能域。
2大黄素和维生素C对团头鲂生长、生理生化指标以及两种HSP70s mRNA表达的影响
饲养试验结束后,测定团头鲂的生长性能,并采样检测鱼的肌肉成分、血液和肝脏生化指标以及肝脏两种HSP70s mRNA的表达水平。结果表明,与对照组相比,大黄素、Vc组显著提高了鱼体增重率和特定生长率,血清中总蛋白(TP)、溶菌酶(LSZ)和碱性磷酸酶(AKP)的水平,肝脏中超氧化物歧化酶(SOD)的活性和诱导型HSP70 mRNA的基础表达水平,降低了饵料系数、死亡率、血清中皮质醇(COR)、甘油三酯(TG)以及肝脏丙二醛(MDA)的含量(P<0.05)；配伍组虽然血清中TP、LSZ的含量以及肝脏HSP70 mRNA的水平显著升高,肝脏MDA的含量也显著降低(P<0.05),但均未表现出协同增效作用。此外,与对照组相比,大黄素组还显著提高了团头鲂肝脏过氧化氢酶(CAT)的活性(P<0.05),而Vc和配伍组CAT的活性与对照组的差异不显著(P>0.05)；其它一些指标如血清中葡萄糖(GLu)、谷丙转氨酶(GPT)、谷草转氨酶(GOT)、胆固醇(CHOL)、肝脏组成型HSC70 mRNA的表达水平、鱼体形体指标以及肌肉组成成分等各组之间差异均不显著(P>0.05)。
3大黄素和维生素C对团头鲂在拥挤胁迫下的生化指标及两种HSP70s mRNA表达的影响
饲养试验结束后,从各池中取25尾规格基本一致的团头鲂,进行连续48 h的拥挤胁迫(100g/L)试验,分别于0h、12h、24h、48h取样分析团头鲂血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平,并统计各组鱼的累积死亡率。结果表明,在拥挤胁迫后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清中TP和AKP的水平,肝脏中SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清中COR、GLU、GPT、GOT、TG以及肝脏MDA的水平,而LSZ的活性表现为先下降后升高；配伍组中,这些指标虽然有类似以上的变化趋势,但大多差异不显著(P>0.05),且同样未表现出协同增效作用。此外,各组鱼血清CHOL的水平在拥挤胁迫前后的差异均不显著(P>0.05)。统计表明,大黄素和Vc组鱼的累积死亡率在拥挤胁迫24h、48h均显著低于对照组(P<0.05),而配伍组与对照组的差异均不显著(P>0.05)。
4大黄素和维生素C对团头鲂在高温应激下的生化指标及两种HSP70s mRNA表达的影响
饲养试验结束后,从各池中取25尾规格基本一致的鱼进行高温应激(34℃)试验。取样检测团头鲂在高温应激前后血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平,并统计各组鱼在高温应激下的累积死亡率。结果表明,在高温应激后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清TP、LSZ和AKP的水平,肝脏SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清COR、GLU、GPT、GOT、TG以及肝脏MDA的水平；配伍组中,这些指标虽然也有类似以上的变化趋势,但大多差异不显著(P>0.05),且未表现出协同增效作用。此外,各组鱼血清胆固醇的含量在高温应激前后的差异均不显著(P>0.05)。统计表明,在高温应激下,Vc组鱼的累积死亡率均显著低于对照组,大黄素组鱼的累积死亡率除6h外,也显著低于对照组(P<0.05),而配伍组与对照组的差异均不显著(P>0.05)。
5大黄素和维生素C对团头鲂感染嗜水气单胞菌后的生化指标及两种HSP70s mRNA表达的影响
饲养试验结束后,从各池中取15尾团头鲂进行腹腔注射感染嗜水气单胞菌试验,并在攻毒前后采样检测团头鲂血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平；另从各池中取10尾鱼进行同样的攻毒试验,并统计攻毒后鱼的累积死亡率。结果表明,经攻毒后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清TP.LSZ和AKP的水平,肝脏SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清COR、GLU、GPT、GOT、TG以及肝脏MDA的水平；配伍组中,这些指标虽然有类似上述的变化趋势,但大多与对照组的差异不显著(P>0.05),且未表现出协同增效作用。此外,各组鱼血清胆固醇的含量在攻毒前后的差异均不显著(P>0.05)。统计也表明,经攻毒后,大黄素和Vc组鱼的累积死亡率在24 h、48 h均显著低于对照组(P<0.05),而配伍组鱼的累积死亡率与对照组的差异均不显著(P>0.05)。
6团头鲂两种热休克蛋白70的原核表达、纯化及鉴定
采用PCR方法扩增团头鲂组成型HSC70和诱导型HSP70基因完整的编码区片段,并分别克隆到原核表达载体pET-22b(+)中,经双酶切和DNA测序鉴定后,将这两种重组表达质粒分别转化大肠杆菌BL21 (DE3),用1 mmol/L IPTG在不同温度及时间下进行诱导表达,并对表达产物进行SDS-PAGE分析,建立最佳诱导表达条件。采用Ni-NTA His Bind Resins亲和层析和DEAE-Sepharose FF阴离子交换柱层析对目的蛋白进行纯化,并进行Western-blotting验证。结果表明,成功构建了团头鲂两种重组表达质粒pET-22b(+)/Ma-HSC70和pET-22b(+)/Ma-HSP70,表达融合蛋白的相对分子量均约为72 kD,并能与兔抗人HSP70多抗进行特异性结合,这两种HSP70s融合蛋白经纯化后的纯度均达到95%以上。在较高温度(37℃)下,有利于融合蛋白的快速大量表达,但易形成包涵体；在较低温度(25℃)下,有利于融合蛋白的可溶性表达,但也会降低融合蛋白的表达速度。综合各种因素,本实验选择融合蛋白Ma-HSC70在25℃和Ma-HSP70在30℃下分别诱导7h作为可溶性表达的最佳条件。
总之,本研究首次从团头鲂肝脏中克隆到两种HSP70s基因,并对该基因的结构和表达特征进行了分析,确定Ma-HSC70基因为组成型,而Ma-HSP70基因为诱导型。在此基础上成功构建了团头鲂两种HSP70s的原核表达系统,并优化了表达条件,最后得到了纯度在95%以上的两种热休克蛋白70的融合蛋白。这些为进一步研究这两种蛋白的结构和功能以及相应抗体的制备打下了基础。饲养和应激试验则表明,在基础日粮中添加大黄素60mg/kg或Vc 700 mg/kg可促进团头鲂的生长,降低饵料系数,提高鱼体的非特异性免疫力、抗氧化和抗应激能力,减少应激死亡率。二者配伍则效果不佳,其相互作用的机理还有待于进一步的研究。
Emodin is an active anthraquinone constituent extracted from the rhizome of rhubarb Rheum officinale Baill. It has many functions such as anti-bacterial and anti-inflammatory, antivirus, antioxidation and scavenging free radicals, reducing blood lipid, protecting the liver, and regulating immunity. Vitamin C (Vc), also known as L-ascorbic acid, is an essential nutrient for normal physiological functions in fish. Vc is an important immunostimulant and antioxidant. but most teleosts are unable to synthesize ascorbic acid due to lack of L-gulonolactone oxidase (EC 1.1.3.8) responsible for the de novo synthesis of Vc. Therefore, exogenous Vc is required in fish diet. In order to study the effects of dietary emodin, high-dose Vc and their combination on growth, non-specific immunity and anti-stress of fish, Wuchang bream (Megalobrama amblycephala, Yih), one of the major freshwater aquaculture fish, was selected as the research subject. Firstly, two full-length cDNA sequences of two HSP70s genes were isolated and cloned from the liver of Wuchang bream, and their gene structure and expression characteristics were analyzed. Then, two HSP70s prokaryotic expression systems were constructed on the basis of above two HSP70s genes, and the expression conditions were optimized. The expressed products were purified and identified. At the same time,1200 healthy fish with initial body weight of (133.44±2.11) g were selected and randomly divided into four groups:a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin,700 mg/kg Vc, and the combination of 60 mg/kg emodin+700 mg/kg Vc, respectively. Each diet was tested in triplicate. After continuous feeding for 60 days, the rearing experiment was over. Then, the experiments of crowding stress, heat stress and infected with Aeromonas hydrophila were carried out.
1 Molecular cloning and sequence analysis of two HSP70 genes in Wuchang bream
Two full-length cDNA sequences of two HSP70s genes were isolated from the liver of Wuchang bream using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70 genes, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly(A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kD, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule, belonging to the constitutive HSC70 whereas Ma-HSP70 gene did not contain any intron in the coding region, belonging to the inducible HSP70. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinaseⅡphosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins wereα-helix and random coil, and their spatial structures included N-terminal ATPase domain and C-terminal polypeptide-binding domain.
2 Effects of emodin and vitamin C on growth, physiological and biochemical parameters, and two HSP70s mRNA expression of Wuchang bream
At the end of the rearing experiment, the growth performance, muscle composition, biochemical parameters of serum and liver, and the mRNA levels of liver two HSP70s genes were investigated. The results showed that compared with the control, the weight gain and specific growth rate, the levels of total protein (TP), lysozyme (LSZ) and alkaline phosphatase (AKP) in serum, the activity of superoxide dismutase (SOD) and basal expression level of inducible HSP70 mRNA in liver significantly increased (P<0.05) while feed coefficient, mortality, the contents of serum cortisol (COR) and triglycerides (TG), and liver malondialdehyde (MDA) decreased (P<0.05) in emodin and Vc groups. Although serum TP, LSZ levels, and the mRNA level of liver HSP70 gene increased significantly (P <0.05), and liver MDA content also significantly decreased (P<0.05) in combination group, the synergism didn't appear. In addition, liver catalase (CAT) activity was also significantly improved in emodin group compared with the control (P<0.05) while the CAT activities in Vc and combination groups were not significantly different with the control (P> 0.05). The other indices, such as serum glucose (GLU), glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), cholesterol (CHOL), the mRNA level of liver constitutive HSC70, the fish body indices, and muscle composition were not significantly different among all groups (P> 0.05).
3 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream under crowding stress
At the end of the rearing experiment,25 fish per tank were exposed to crowding stress (100 g/L) for 48 h, the biochemical parameters of blood and liver, mRNA levels of liver two HSP70s genes, and cumulative mortalities of fish in each group were investigated at 0 h,12 h,24 h and 48 h, respectively. The results showed that, after crowding stress, compared with the control, serum TP and AKP levels, liver SOD and CAT activities, and the expression levels of liver HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees, and serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent, while the LSZ activity decreased at first and then increased significantly (P<0.05). In combination group, although these parameters had similar changing trend as above ones, most of them were not significant differences with the control(P>0.05), and did not show any synergism either. In addition, serum CHOL levels among all groups before and after crowding stress were not significantly different (P> 0.05). The statistics showed that, the cumulative mortalities of the emodin and Vc groups were significantly lower than that of the control at 24 h and 48 h (P<0.05), but there was no significant difference between the combination and control groups (P> 0.05).
4 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream under high temperature stress
At the end of the rearing experiment,25 fish per tank were exposed to high temperature stress of 34℃. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that, after heat stress, compared with the control, serum TP, LSZ, and AKP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. In addition, serum cholesterol levels among all groups before and after heat stress were not significantly different (P>0.05). Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P<0.05) while the difference between the combination and control groups was not significant (P>0.05).
5 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream infected with Aeromonas hydrophila
At the end of the rearing experiment,15 fish per tank were intraperitoneally infected with A. hydrophila (BSK-10), and the biochemical parameters of serum and liver, the mRNA expression levels of liver two HSP70s genes before and after infection were determined. Another ten fish taken from each tank were challenged with A. hydrophila as the same method as above, the cumulative mortality of each group was counted at different time point of post-infection. The results showed that after infection of fish, compared with the control, serum TP, LSZ and AKP levels, liver SOD, CAT activities, and the expression levels of HSC70 and HSP70 mRNA increased in emodin and Vc groups in varying degrees, while serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent. In combination group, although these parameters had similar changing trend as above ones, most of them were not significant differences with the control(P>0.05), and did not show any synergism either. In addition, serum cholesterol levels among all groups before and after infection were not significantly different (P>0.05). Statistics also showed that after infected with A. hydrophila, the cumulative mortalities of emodin and Vc groups were significantly lower than that of the control in 24 h and 48 h (P<0.05), but the difference of the cumulative mortality between the combination and control groups was not significant (P>0.05).
6 Prokaryotic expression, purification and identification of two HSP70s in Wuchang bream
The fragments of constitutive HSC70 and inducible HSP70 ORFs of Wuchang bream were amplified by PCR, and then cloned into prokaryotic expression vectors of pET-22b(+), respectively. The two recombinant plasmids, determined by double-endonuclease digestion and sequencing, were transformed into competent E. coli BL21 (DE3), respectively. The two recombinant strains were induced by 1 mmol/L IPTG at different temperatures and times, establishing optimal conditions for inducible expression. The expression products were purified by Ni-NTA His Bind Resins affinity chromatography and DEAE-Sepharose FF anion-exchange chromatography, and detected by SDS-PAGE and Western-blotting, respectively. The results show that two recombinant expression plasmids of pET-22b(+)/Ma-HSC70 and pET-22b(+)/Ma-HSP70, each expressing a fusion protein of about 72 kD identified by rabbit source anti-HSP70 polyclonal antibody, were successfully constructed, and the purity of two purified fusion proteins was more than 95%. A higher temperature (37℃) is conducive to the rapid expression of fusion protein, but it is easy to form inclusion body. A lower temperature (25℃) is in favor of the soluble expression of fusion protein, but it could also reduce the expression rate of fusion protein. All things considered, the optimal conditions for soluble expression of the two fusion proteins of Ma-HSC70 and Ma-HSP70 are induced for 7 h at 25℃and 30℃, respectively.
In conclusion, two full-length HSP70 cDNAs were cloned and characterized from Wuchang bream liver for the first time. There are seven introns in the coding region of Ma-HSC70 gene while there is no intron in the Ma-HSP70 gene, suggesting constitutive and inducible expression, respectively. Through optimizing the expression conditions, two purified fusion proteins of Ma-HSC70 and Ma-HSP70, which purities were more than 95%, have been obtained by prokaryotic expression systems of two HSP70s genes of Wuchang bream. Thus it laid a foundation for further study on molecular structure and function of the two proteins and associated antibodies. The rearing and stress experiments indicate that, the basal diet supplemented with either 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, lower FCR, increase non-specific immunity, antioxidant capacity, and stress resistance, reduce stress mortality. However, the combination of emodin and high-dose Vc showed no better effects. The mechanism of their interaction needs to be further studied.
引文
1. Ackerman P A, Iwama G K. Physiological and cellular stress responses of juvenile rainbow trout to vibriosis [J]. J Aquat Anim Health,2001,13(2):173-180
2. Affonso E G, Silva Eda C, Tavares-Dias M, et al. Effect of high levels of dietary vitamin C on the blood responses of matrinxa (Brycon amazonicus) [J]. Comp Biochem Physiol A,2007,147(2): 383-388
3. Ai Q H, Mai K S, Tan B P, et al. Effects of dietary vitamin C on survival, growth, and immunity of large yellow croaker, Pseudosciaena crocea [J]. Aquaculture,2006,261(1):327-336
4. Airaksinen S, Jokilehto T, Rabergh C M I, et al. Heat-and cold-inducible regulation of HSP70 expression in zebrafish ZF4 cells [J]. Comp Biochem Physiol B,2003,136(2):275-282
5. Ali K S, Dorgai L, Abraham M, et al. Tissue-and stressor-specific differential expression of two hsc70 genes in carp [J]. Biochem Biophys Res Commun,2003,307(3):503-509
6. Alves D S, Perez-Fons L, Estepa A, et al. Membrane-related effects underlying the biological activity of the anthraquinones emodin and barbaloin [J]. Biochem Pharmacol,2004,68(3):549-561
7. Amin J, Fernandez M, Ananthan J, et al. Cooperative binding of heat shock transcription factor to the Hsp70 promoter in vivo and in vitro [J]. Biol Chem,1994,269(7):4804-4811
8. Anderson D P, Dixon O W. Suppression of antibody-producing cells in rainbow trout spleen sections exposed to copper in vitro [J]. J Aquat Anim Health,1989,1(1):57-61
9. Anderson D P. Immunostimulants, adjuvants and vaccine carrier in fish:application to aquaculture [J]. Annu Rev Fish Dis,1992,2:281-307
10. Anderson I, Brass A. Searching DNA databases for similarities to DNA sequences:when is a match significant? [J] Bioinformatics,1998,14(4):349-356
11. Angelidis C E, Lazaridis I, Pagoulatos G N. Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance [J]. Eur J Biochem,1991,199(1):35-39
12. Ansary A, Haneef R M, Torres J L, et al. Plasmids and antibiotic resistance in Aeromonas hydrophila isolated in Malaysia from healthy and diseased fish [J]. J Fish Dis,1992,15(2):191-196
13. Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL workspace:a web-based environment for protein structure homology modeling [J]. Bioinformatics,2006,22(2):195-201
14. Baler R, Dahl G, Voellmy R. Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1 [J]. Mol Cell Biol,1993,13(4):2486-2496
15. Barnett C W, Pankhurst N W. The effects of common laboratory and husbandry practices on the stress response of greenback flounder Rhombosoleu tapirina (Giinther,1862) [J]. Aquaculture, 1998,162(3-4):313-329
16. Barton B A, Iwama G K. Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids [J]. Annual Rev Fish Dis,1991,1:3-26
17. Basu N, Todgham A E, Ackerman P A, et al. Heat shock protein genes and their functional significance in fish [J]. Gene,2002,295(2):173-183
18. Bayne C J, Gerwick L. The acute phase response and innate immunity of fish [J]. Dev Comp Immunol,2001,25(8-9):725-743
19. Beere H M, Wolf B B, Cain K, et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome [J]. Nat Cell Biol,2000,2(8):469-475
20. Bernelli-Zazzera A, Cairo G, Schiaffonati L, et al. Stress proteins and reperfusion stress in the liver [J]. Ann N Y Acad of Sci,1992,663:120-124
21. Bernier N J, Peter R E. The hypothalamic-pituitary-interrenal axis and the control of food intake in teleost fish [J]. Comp Biochem and Physiol B,2001,129(2-3):639-644
22. Blom J H, Dabrowski K. Ascorbic acid metabolism in fish:is there a maternal effect on the progeny? [J]. Aquaculture,1996,147(3-4):215-224
23. Boone A N, Vijayan M M. Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes:effect of heat shock and heavy metal exposure [J]. Comp Biochem Physiol C,2002, 132(2):223-233
24. Bork P, Sander C, Valencia A. An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins [J]. Proc Natl Acad Sci USA,1992,89(16): 7290-7294
25. Boutet I, Tanguy A, Rousseau S, et al. Molecualr identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas [J]. Cell Stress Chaperones,2003,8 (1):76-85
26. Breathnach R, Chambon P. Organization and expression of eucaryotic split genes coding for proteins [J]. Annu Rev Biochem,1981,50:349-383
27. Brock P, Eldred E W, Woiszwillo J E, et al. Direct solid-phase 1251 radioimmunoassy of serum cortisol [J]. Clin Chem,1978,24(9):1595-1598
28. Brown S B, MacLatchy D L, Hara T J, et al. Effects of ambient pH and aluminum on plasma kinetics of cortisol, T3, and T4 in rainbow trout(Oncorhynchus mykiss) [J]. Can J Zool,1990,68(7): 1537-1543
29. Burdon R H. Heat shock and the heat shock proteins [J]. Biochem J,1986,240(2):313-324
30. Burdon R H. Heat shock proteins in relation to medicine [J]. Mol Aspects Med,1993,14(2):83-165
31. Cao Y, Ohwatari N, Matsumoto T, et al. TGF-β1 mediates 70-kDa heat shock protein induction due to ultraviolet irradiation in human skin fibroblasts [J]. Pflugers Arch,1999,438(3):239-244
32. Cara J B, Aluru N, Moyano F J, et al. Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout [J]. Comp Biochem Physiol,2005,142(4): 426-431
33. Castelli C, Ciupitu A M, Rini F, et al. Human heat shock protein 70 peptide complexes specifically activate antimelanoma T cells [J]. Cancer Res,2001,61(1):222-227
34. Chang C H, Lin C C, Yang J J, et al. Anti-inflammatory effects of emodin from ventilago leiocarpa [J]. Am J Chin Med,1996,24(2):139-142
35. Chen R, Lochmann R, Goodwin A, et al. Effects of dietary vitamins C and E on alternative complement activity, hematology, tissue composition, vitamin concentrations and response to heat stress in juvenile golden shiner (Notemigonus crysoleucas) [J]. Aquaculture,2004,242(1-4): 553-569
36. Chen W H, Sun L T, Tsai C L, et al. Cold-stress induced the modulation of catecholamines, cortisol, immunoglobulin M, and leukocyte phagocytosis in tilapia [J]. Gen Comp Endocrinol,2002, 126(1):90-100
37. Cheng P Z, Liu X, Zhang G F, et al. Cloning and expression analysis of a HSP70 gene from pacific abalone (Haliotis discus hannai) [J]. Fish Shellfish Immunol,2007,22(1-2):77-87
38. Chilmonczyk S. Rainbow trout lymphoid organs:cellular effects of corticosteroids and anti-thymocyte serum [J]. Dev Comp Immunol,1982,6(2):271-280
39. Clearwater S J, Pankhurst N W. The response to capture and confinement stress of plasma cortisol, plasma sex steroids and vitellogenic oocytes in the marine teleost, red gurnard [J]. J Fish Biol,1997, 50(2):429-441
40. Clerton P, Troutaud D, Verlhac V, et al. Dietary vitamin E and rainbow trout (Oncorhynchus my kiss) phagocyte functions:effect on gut and on head kidney leucocytes [J]. Fish Shellfish Immunol,2001, 11(1):1-13
41. Collazos M E, Barriga C, Ortega E. Effect of high summer temperatures upon granulocyte phagocytic function of the tench (Tinea tinea, L.) [J]. Comp Immunol Microbiol infect Dis,1995, 18(2):115-121
42. Creagh E M, Carmody R J, Cotter T G. Heat shock protein 70 inhibits caspase-dependent and-independent apoptosis in Jurkat T cells [J]. Exp Cell Res,2000,257(1):58-66
43. Dabrowska H, Dabrowski K, Meyer-Burgdorff K, et al. The effect of large doses of vitamin C and magnesium on stress responses in common carp, Cyprinus carpio [J]. Comp Biochem Physiol A, 1991,99(4):681-685
44. Dabrowski K, Ciereszko A. Ascorbic acid protects against male infertility in a teleost fish [J]. Experientia,1996,52(2):97-100
45. Dang S S, Zhang Z G, Chen Y R, et al. Inhibition of the replication of hepatitis B virus in vitro by emodin [J]. Med Sci Monit,2006,12(9):302-306
46. Das D K, Engelman R M, Kimura Y. Molecular adaptation of cellular defences following preconditioning of the heart by repeated ischaemia [J]. Cardiovasc Res,1993,27(4):578-584
47. Das P, Gupta A, Manna S K. Heat shock protein 70 expression in different tissues of Cirrhinus mrigala (Ham.) following heat stress [J]. Aquae Res,2005,36(6):525-529
48. Dave G, Johansson-Sjobeck M L, Larsson A, et al. Effects of cortisol on the fatty acid composition of the total blood plasma lipids in the European eel, Anguilla anguilla L. [J]. Comp Biochem Physiol A,1979,64(1):37-40
49. de Menezes G C, Tavares-Dias M, Ono E A, et al. The influence of dietary vitamin C and E supplementation on the physiological response of pirarucu, Arapaima gigas, in net culture [J]. Comp Biochem Physiol A,2006,145(2):274-279
50. Deane E E, Li J, Woo N Y. Modulated heat shock protein expression during pathogenic Vibrio alginolyticus stress of sea bream [J]. Dis Aquat Organ,2004,62(3):205-215
51. Deane E E, Woo N Y S. Cloning and characterization of the hsp70 multigene family from silver sea bream:Modulated gene expression between warm and cold temperature acclimation [J]. Biochem Biophys Res Commun,2005,330(3):776-783
52. Demand J, Liiders J, Hohfeld J. The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors [J]. Mol Cell Biol,1998,18(4):2023-2028
53. Demers N E, Bayne C J. The immediate effects of stress on hormones and plasma lysozyme in rainbow trout [J]. Dev Comp Immunol,1997,21(4):363-373
54. Di Marco P, Priori A, Finoia M G, et al. Physiological responses of European sea bass Dicentrarchus labrax to different stocking densities and acute stress challenge [J]. Aquaculture, 2008,275(1-4):319-328
55. DuBeau S F, Pan F, Tremblay G C, et al. Thermal shock of salmon in vivo induces the heat shock protein hsp70 and confers protection against osmotic shock [J]. Aquaculture,1998,168(1-4): 311-323
56. Dziewulska-Szwajkowska D, Lozinska-Gabska M, Adamowicz A, et al. The effect of high dose of cortisol on glucose-6-phosphatase and fructose-1,6-bisphosphatase activity, and glucose and fructose-2,6-bisphosphate concentration in carp tissues (Cyprinus carpio L.) [J]. Comp Biochem Physiol B,2003,135(3):485-491
57. Ellis R J. The molecular chaperone concept [J]. Semin Cell Biol,1990,1(1):1-9
58. Ellsaesser C F, Clem L W. Haematological and immunological changes in channel catfish stressed by handling and transport [J]. J Fish Biol,1986,28(4):511-521
59. Espelid S, Lekken G B, Steiro K, et al. Effects of cortisol and stress on the immune system in Atlantic salmon (Salmo salar L.) [J]. Fish Shellfish Immunol,1996,6(2):95-110
60. Feder M E, Hofmann G E. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology [J]. Annu Rev Physiol,1999,61(1):243-282
61. Fevolden S E, Refstie T, Rφed K. H. Selection for high and low cortisol stress response in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) [J]. Aquaculture,1991,95(1-2): 53-65
62. Fevolden S E, Rφed K H, Fjalestad K. T, et al. Poststress levels of lysozyme and cortisol in adult rainbow trout:heritabilities and genetic correlations [J]. J Fish Biol,1999,54(4):900-910
63. Fevolden S E, Rφed K H, Fjalestad K. A combined salt and confinement stress enhances mortality in rainbow trout (Oncorhynchus mykiss) selected for high stress responsiveness [J]. Aquaculture, 2003,216(1-4):67-76
64. Fevolden S E, Reed K H. Cortisol and immune characteristics in rainbow trout (Oncorhynchus mykiss) selected for high or low tolerance to stress [J]. J Fish Biol,1993,43(6):919-930
65. Fevolden S E, Rφed K H, Fjalestad K. A combined salt and confinement stress enhances mortality in rainbow trout (Oncorhynchus mykiss) selected for high stress responsiveness [J]. Aquaculture, 2003,216(1-4):67-76
66. Foo J T W, Lam T J. Retardation of ovarian growth and depression of serum steroid levels in the tilapia, Oreochromis mossambicus, by cortisol implantation [J]. Aquaculture,1993,115(1-2): 133-143
67. Fracalossi D M, Allen M E, Nichols D K, et al. Oscars, Astronotus ocellatus, have a dietary requirement for vitamin C [J]. J Nutr,1998,128(10):1745-1751
68. Fracalossi D M, Allen M E, Yuyama L K, et al. Ascorbic acid biosynthesis in Amazonian fishes [J]. Aquaculture,2001,192(2-4):321-332
69. Franzellitti S, Fabbri E. Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors [J]. Biochem Biophys Res Commun,2005,336(4):1157-1163
70. Freeman B A, Crapo J D. Biology of disease:free radicals and tissue injury [J]. Lab Invest,1982, 47(5):412-426
71. Freeman B C, Myers M P, Schumacher R, et al. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1 [J]. EMBO J,1995,14(10): 2281-2292
72. Gabai V L, Meriin A B, Mosser D D, et al. Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance [J]. J Biol Chem,1997,272(29):18033-18037
73. Garrido C, Gurbuxani S, Ravagnan L, et al. Heat shock proteins:endogenous modulators of apoptotic cell death [J]. Biochem Biophys Res Commun,2001,286(3):433-442
74. Goel R K, Das Gupta G, Ram S N, et al. Antiulcerogenic and anti-inflammatory effects of emodin, isolated from Rhamnus triquerta wall [J]. Indian J Exp Biol,1991,29(3):230-232
75. Gornati R, Papis E, Rimoldi S, et al. Rearing density influences the expression of stress-related genes in sea bass (Dicentrarchus labrax, L.)[J]. Gene,2004,341:111-118
76. Granelli K, Bjorck L, Appelqvist L A. The variation of superoxide dismutase (SOD) and xanthine oxidase (XO) activities in milk using an improved method to quantitate SOD activity [J]. J Sci Food Agric,1995,67(1):85-91
77. Grutter A S, Pankhurst N W. The effects of capture, handling, confinement and ectoparasite load on plasma levels of cortisol, glucose and lactate in the coral reef fish Hemigymnus melapterus [J]. J Fish Biol,2000,57(2):391-401
78. Gursu M F, Onderci M, Gulcu F, et al. Effects of vitamin C and folic acid supplementation on serum paraoxonase activity and metabolites induced by heat stress in vivo [J]. Nutr Res,2004, 24(2):157-164
79. Guy C L, Li Q B. The organization and evolution of the spinach stress 70 molecular chaperone gene family [J]. Plant Cell,1998,10(4):539-556
80. Haddy J A, Pankhurst N W. Stress-induced changes in concentrations of plasma sex steroids in black bream [J]. J Fish Biol,1999,55(6):1304-1316
81. Hall T J. Role of hsp70 in cytokine production [J]. Cell Mol Life Sci,1994,50(11-12):1048-1053
82. Hardie L J, Fletcher T C, Secombes C J. The effect of dietary vitamin C on the immune response of the Atlantic salmon (Salmo solar L.) [J]. Aquaculture,1991,95(3-4):201-214
83. Harrison C J, Bohm A A, Nelson H C. Crystal structure of the DNA binding domain of the heat shock transcription factor [J]. Science,1994,263(5144):224-227
84. Hartl F U, Hayer-Hartl M. Molecular chaperones in the cytosol:from nascent chain to folded protein [J]. Science,2002,295(5561):1852-1858
85. Hartl F U. Molecular chaperones in cellular protein folding [J]. Nature,1996,381(6583):571-579
86. Haug T, Kjuul A K, Stensvag K, et al. Antibacterial activity in four marine crustacean decapods [J].Fish Shellfish Immunol,2002,12(5):371-385
87. Helenius A, Aebi M. Intracellular functions of N-linked glycans [J]. Science,2001,291(5512): 2364-2369
88. Hendrick J P, Hartl F U. Molecular chaperone functions of heat-shock proteins [J]. Annu Rev Biochem,1993,62:349-384
89. Henrique M M F, Gomes E F, Gouillou-Coustans M F, et al. Infulence of supplementation of practical diets with vitamin C on growth and response to hypoxic stress of seabream, Sparus aurata [J]. Aquaculture,1998,161(1-4):415-426
90. Hightower L E. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides [J]. J Cell Physiol,1980,102(3):407-427
91. Hightower L E. Heat shock, stress proteins, chaperones, and proteotoxicity [J]. Cell,1991,66(2): 191-197
92. Hofmann G E, Buckley B A, Airaksinen S, et al. Heat-shock protein expression is absent in the Antarctic fish Trematomus bernacchii (family Nototheniidae) [J]. J Exp Biol,2000,203:2331-2339
93. Hrubec T C, Robertson J L, Smith S A, et al. The effect of temperature and water quality on antibody response to Aeromonas salmonicida in sunshine bass (Morone chrysops× Morone saxatilis) [J]. Vet Immunol Immunopathol,1996,50(1-2):157-166
94. Hsieh S L, Chen Y N, Kuo C M. Physiological responses, desaturase activity, and fatty acid composition in milkfish (Chanos chanos) under cold acclimation [J]. Aquaculture,2003,220(1-4): 903-918
95. Huang S S, Yeh S F, Hong C Y. Effect of anthraquinone derivatives on lipid peroxidation in rat heart mitochondria:structure activity relationship [J]. J Nat Prod,1995,58(9):1365-1371
96. Huang Y, Wimler K M, Carmichael G G. Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing [J]. EMBO J,1999,18(6):1642-1652
97. Hwang D F, Lin T K. Effect of temperature on dietary vitamin C requirement and lipid in common carp [J]. Comp Biochem Physiol B,2002,131(1):1-7
98. Iguchi K, Ogawa K, Nagae M, et al. The influence of rearing density on stress response and disease susceptibility of ayu (Plecoglossus altivelis) [J]. Aquaculture,2003,220(1-4):515-523
99. Ishibashi Y, Kato K, Ikeda S, et al. Effects of dietary ascorbic acid on tolerance to intermittent hypoxic stress in Japanese parrot fish [J]. Nippon Suisan Gakkaishi,1992,58(11):2147-2152
100. Ishioka H. Live Fish Transportation, Series of Fisheries Book 39 [M]. Tokyo:Kosesha Kosekaku, 1982:52-69
101. Israeli D, Kimmel E. Monitoring the behavior of hypoxia-stressed Carassius auratus using computer vision [J]. Aquacult Eng,1996,15(6):423-440
102. Israeli-Weinstein D, Kimmel E. Behavioral response of carp (Cyprinus carpio) to ammonia stress [J]. Aquaculture,1998,165(1-2):81-93
103. Ito A, Fujioka M, Tanaka K, et al. Screening of cytokines to enhance vaccine effects of heat shock protein 70-rich tumor cell lysate [J]. J Biosci Bioeng,2005,100(1):36-42
104. Iwama G K, Vijayan M M, Forsyth R B, et al. Heat shock proteins and physiological stress in fish [J]. Amer Zool,1999,39(6):901-909
105. Janssens P A, Waterman J. Hormonal regulation of gluconeogenesis and glycogenolysis in carp (Cyprinus carpio) liver pieces cultured in vitro [J]. Comp Biochem Physiol A,1988,91(3):451-455
106. Jayasuriya H, Koonchanok N M, Geahlen R L, et al. Emodin, a protein tyrosine kinase inhibitor from Polygonum cuspidatum [J]. J Nat Prod,1992,55(5):696-698
107. Jeney G, Galeotti M, Volpatti D, et al. Prevention of stress in rainbow trout(Oncorhynchus mykiss) fed diets containing different doses of glucan [J]. Aquaculture,1997,154(1):1-15
108. Jiao C Z, Wang Z Z, Li F H, et al. Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis [J]. Chin Sci Bull,2004, 49(22):2385-2393
109. Jing X, Ueki N, Cheng J, et al. Induction of apoptosis in hepatocellular carcinoma cell lines by emodin [J]. Jpn J Cancer Res,2002,93(8):874-882
110. Johnston R N, Kucey B L. Competitive inhibition of hsp70 gene expression causes thermosensitivity [J]. Science,1988,242(4885):1551-1554
111. Johnston W L, MacDonald E, Hilton J W. Relationships between dietary ascorbic acid status and deficiency, weight gain and brain neurotransmitter levels in juvenile rainbow trout, Salmo gairdneri [J]. Fish Physiol Biochem,1989,6(6):353-365
112. Kanazawa A. Effects of docosahexaenoic acid and phospholipids on stress tolerance of fish [J]. Aqauculture,1997,155(1-4):129-134
113. Khassaf M, McArdle A, Esanu C, et al. Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle [J]. J Physiol,2003,549(2):645-652
114. Kiang J G, Tsokos G C. Heat shock protein 70 kDa:molecular biology, biochemistry and physiology [J]. Pharmacol Ther,1998,80(2):183-201
115. Kitabchi A E. Ascorbic acid in steroidogenesis [J]. Nature,1967,215:1385-1386
116. Knowlton A A, Salfity M. Nuclear localization and the heat shock proteins [J]. J Biosci,1996, 21(2):123-132
117. Kohler M, Haller H, Hartmann E. Nuclear protein transport pathways [J]. Exp Nephrol,1999,7(4): 290-294
118. Kothary R K, Jones D, Candido E P M.70-Kilodalton heat shock polypeptides from rainbow trout: characterization of cDNA sequences [J]. Mol Cell Biol,1984,4(9):1785-1791
119. Koven W, Barr Y, Lutzky S, et al. The effect of dietary arachidonic acid (20:4n-6) on growth, survival and resistance to handling stress in gilthead seabream (Sparus aurata) larvae [J]. Aquaculture,2001,193(1-2):107-122
120. Kregel K C. Heat shock proteins:modifying factors in physiological stress responses and acquired thermotolerance [J]. J Appl Physiol,2002,92(5):2177-2186
121. Kroeger P E, Sarge K D, Morimoto R I. Mouse heat shock transcription factors 1 and 2 prefer a trimeric binding site but interact differently with the HSP70 heat shock element [J]. Mol and Cell Biol,1993,13(6):3370-3383
122. Kroemer G, Reed J C. Mitochondrial control of cell death [J]. Nat Med,2000,6(5):513-519
123. Kuhn E R, Geris K L, van der Geyten S, et al. Inhibition and activation of the thyroidal axis by the adrenal axis in vertebrates [J]. Comp Biochem Physiol A,1998,120(1):169-174
124. Kumari J, Sahoo P K. High dietary vitamin C affects growth, non-specific immune responses and disease resistance in Asian catfish, Clarias batrachus [J]. Mol Cell Biochem,2005,280(1-2):25-33
125. Landolt M L. The relationship betwen diet and the immune response of fish [J]. Aquaculture,1989, 79(1-4):193-206
126. Le Morvan C, Deschaux P, Troutaud D. Effects and mechanisms of environmental temperature on carp (Cyprinus carpio) anti-DNP antibody response and non-specific cytotoxic cell activity:a kinetic study [J]. Dev Comp Immunol,1996,20(5):331-340
127. Lee H Z. Protein kinase C involvement in aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell [J]. Br J Pharmacol,2001,134(5):1093-1103
128. Lewis J, Devin A, Miller A, et al. Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-κB activation [J]. J Biol Chem,2000,275(14):10519-10526
129. Li M H, Wise D J, Robinson E H. Effect of dietary vitamin C on weight gain, tissue ascorbate concentration, stress response, and disease resistance of Channel catfish Ictalurus punctatus [J]. J World Aquacult Soc,1998,29(1):1-8
130. Lim C, Lovell R T. Pathology of the vitamin C deficiency syndrome in channel catfish (Ictalurus punctatus) [J]. J Nutr,1978,108(7):1137-1146
131. Lim L C, Dhert P, Chew W Y, et al. Enhancement of stress resistance of the Guppy Poecilia reticulata through feeding with vitamin C supplement [J]. J World Aquacul Soc,2002,33(1):32-40
132. Lin C C, Chang C H, Yang J J, et al. Hepatoprotective effects of emodin from Ventilago Leiocarpa [J].J Ethnopharmacol,1996,52(2):107-111
133. Lin M F, Shiau S Y. Dietary L-ascorbic acid affects growth, nonspecific immune responses and disease resistance in juvenile grouper, Epinephelus malabaricus [J]. Aquaculture,2005,244(1-4): 215-221
134. Lindguist S, Craig E A. The heat-shock proteins [J]. Annu Rev Genet,1988,22:631-677
135. Lis J, Wu C. Heat shock factor [C]//. McKnight S L, Yamamoto K R. Transcriptional Regulation. New York:Cold Spring Harbor Laboratory,1992:907-930
136. Liu B, Xie J, Ge X P, et al. Effects of anthraquinone extract from Rheum officinale Baill on the growth performance and physiological responses of Macrobrachium rosenbergii under high temperature stress [J]. Fish Shellfish Immunol,2010,29(1):49-57
137. Liu J, Yang W J, Zhu X J, et al. Molecular cloning and expression of two HSP70 genes in the prawn, Macrobrachi um rosenbergii [J]. Cell Stress Chaperones,2004,9(3):313-323
138. Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method [J]. Methods,2001,25(4):402-408
139. Lowry O H, Rosebrough N J, Farr A L, et al. Protein measurement with the folin phenol reagent [J]. J Biol Chem,1951,193(1):265-275
140. Macconnell E, Barrows F T. Pathological changes associated with vitamin C deficiency in walleyes [J]. J Aquat Anim Health,1993,5(4):287-293
141.Mackett D B, Tam W H, Fryer J N. Histological changes in insulin-immunoreactive pancreatic β-cell, and suppression of insulin secretion and somatotrope activity in brook trout(Salvelinus fontinalis) maintained on reduced food intake or exposed to acidic environment [J]. Fish Physiol Biochem,1992,10(3):229-243
142. Mahmoud K Z, Edens F W, Eisen E J, et al. Ascorbic acid decreases heat shock protein 70 and plasma corticosterone response in broilers(Gallus gallus domesticus) subjected to cyclic heat stress [J]. Comp Biochem Physiol B,2004,137(1):35-42
143. Manojlovic N T, Solujic S, Sukdolak S, et al. Antifungal activity of Rubia tinctorum, Rhamnus frangula and Caloplaca cerina [J]. Fitoterapia,2005,76(2):244-246
144. Mazik P M, Tomasso J R, Brandt T M. Efects of dietary vitamin C on growth, caudal fin development, and tolerance of aquaculture-related stressors in channel catfish [J]. Prog Fish-Cult, 1987,49(1):13-16
145. Mazur C F, Iwama G K. Handling and crowding stress reduces the number of plaque-forming cells in Atlantic salmon [J]. J Aquat Anim Health,1993,5(2):98-101
146. Mbwambo Z H, Apers S, Moshi M J, et al. Anthranoid compounds with antiprotozoal activity from Vismia orientalis [J]. Planta Med,2004,70(8):706-710
147. McCormick S D, Shrimptom J M, Carey J B, et al. Repeated acute stress reduces growth rate of Atlantic salmon parr and alters plasma levels of growth hormone, insulin-like growth factor I and cortisol [J]. Aquaculture,1998,168(1-4):221-235
148. McCully J D, Myrmel T, Lotz M M, et al. The rapid expression of myocardial hsp 70 mRNA and the heat shock 70 kDa protein can be achieved after only a brief period of retrograde hyperthermic perfusion [J]. J Mol Cell Cardiol,1995,27(3):873-882
149. McGarry T J, Lindquist S. The preferential translation of Drosophila hsp70 mRNA requires sequences in the untranslated leader [J]. Cell,1985,42(3):903-911
150. McRae M P. Vitamin C supplementation lowers serum low-density lipoprotein cholesterol and triglycerides:a meta-analysis of 13 randomized controlled trials [J].J Chiropr Med,2008,7(2): 48-58
151.Mehlen P, Kretz-Remy C, Preville X, et al. Human hsp27, Drosophila hsp27 and human aB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFa-induced cell death [J]. EMBO J,1996,15(11):2695-2706
152. Meng K W, Lv Y, Yu L, et al. Effects of emodin and double blood supplies on liver regeneration of reduced size graft liver in rat model [J]. World J Gastroenterol,2005,11(19):2941-2944
153. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test [J]. Anal Biochem,1978,86(1):271-278
154. Milarski K L, Morimoto R I. Expression of human HSP70 during the synthetic phase of the cell cycle [J]. Proc Natl Acad Sci USA,1986,83(24):9517-9521
155. Ming J H, Xie J, Xu P, et al. Molecular cloning and expression of two HSP70 genes in the Wuchang bream (Megalobrama amblycephala Yih) [J]. Fish Shellfish Immunol,2010,28(3): 407-418
156. Mock A, Peters G. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution [J]. J Fish Biol,1990,37(6):873-885
157. Molina A, Biemar F, Muller F, et al. Cloning and expression analysis of an inducible HSP70 gene from tilapia fish [J]. FEBS Lett,2000,474(1):5-10
158. Mommsen T P, Vijayan M M, Moon T W. Cortisol in teleosts:dynamics, mechanisms of action, and metabolic regulation [J]. Rev Fish Biol Fisher,1999,9(3):211-268
159. Montero D, Marrero M, Izquierdo M S, et al. Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress [J]. Aquaculture,1999,171(3-4):269-278
160. Montero D, Tort L, Robaina L, et al. Low vitamin E in diet reduces stress resistance of gilthead seabream (Sparus aurata) juveniles [J]. Fish Shellfish Immunol,2001,11(6):473-490
161. Morimoto R I, Kroeger P E, Cotto J J. The transcriptional regulation of heat shock genes:a plethora of heat shock factors and regulatory conditions [J]. EXS,1996,77:139-163
162. Morimoto R I. Cells in stress:transcriptional activation of heat shock genes [J]. Science,1993, 259(5100):1409-1410
163. Mosser D D, Caron A W, Bourget L, et al. The chaperone function of hsp70 is required for protection against stress-induced apoptosis [J]. Mol Cell Biol,2000,20(19):7146-7159
164. Nakai A, Tanabe M, Kawazoe Y, et al. HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator [J]. Mol Cell Biol,1997,17(1):469-481
165. Navarre O, Halver J E. Disease resistance and humoral antibody production in rainbow trout fed high levels of vitamin C [J]. Aquaculture,1989,79(1-4):207-221
166. Ndong D, Chen Y Y, Lin Y H, et al. The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures [J]. Fish Shellfish Immunol,2007,22(6):686-694
167. Ng T B, Liu F, Lu Y, et al. Antioxidant activity of compounds from the medicinal herb Aster tataricus [J]. Comp Biochem Physiol C,2003,136(2):109-115
168. Niedzwiecki A, Reveillaud I, Fleming J E. Changes in superoxide dismutase and catalase in aging heat-shocked Drosophila [J]. Free Radic Res,1992,17(6):355-367
169. Ohta T. Further examples of evolution by gene duplication revealed through DNA sequence comparisons [J]. Genetics,1994,138(4):1331-1337
170. Ojolick E J, Cusack R, Benfey T J, et al. Survival and growth of all-female diploid and triploid rainbow trout (Oncorhynchus mykiss) reared at chronic high temperature [J]. Aquaculture,1995, 131(3-4):177-187
171. Olsvik P A, Lie K K, Jordal A E, et al. Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon [J]. BMC Mol Biol,2005,6:21
172. Ortuno J, Esteban M A, Meseguer J. Effect of high dietary intake of vitamin C on non-specific immune response of gilthead seabream (Sparus aurata L.) [J]. Fish Shellfish Immunol,1999,9(5): 429-443
173. Ortuno J, Esteban M A, Meseguer J. Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L.) innate immune response [J]. Fish Shellfish Immunol,2001,11(2):187-197
174. Ozrurk-Urek R, Bozkaya L A, Tarhan L. The effects of some antioxidant vitamin- and trace element-supplemented diets on activities of SOD, CAT, GSH-Px and LPO levels in chicken tissues [J]. Cell Biochem Funct,2001,19(2):125-132
175. Pankhurst N W, Van Der Kraak G, Peter R E. Evidence that the inhibitory effects of stress on reproduction in teleost fish are not mediated by the action of cortisol on ovarian steroidogenesis [J]. Gen Comp Endocrinol,1995,99(3):249-257
176. Pankhurst N W, Van Der Kraak G. Effects of stress on reproduction and growth of fish [C]//Iwama G K, Pickering A D, Sumpter J P, et al. Fish Stres and Health in Aquaculture. Society for Experimental Biology Seminar Series 62. Cambrige, UK:Cambrige Univ Pres,1997:73-93
177. Pankhurst N W, Van Der Kraak G. Evidence that acute stress inhibits ovarian steroidogenesis in rainbow trout in vivo, through the action of cortisol [J]. Gen Comp Endocrinol,2000,117(2): 225-237
178. Paolucci M, Esposito V, di Fiore M M, et al. Effects of short postcapture confinement on plasma reproductive hormone and corticosterone profiles in Rana esculenta during the sexual cycle [J]. Boll Zool,1990,57(3):253-259
179. Parihar M S, Dubey A K, Javeri T, et al. Changes in lipid peroxidation, superoxide dismutase activity, ascorbic acid and phospholipid content in liver of freshwater catfish Heteropneustes fossilis exposed to elevated temperature [J]. J Therm Biol,1996,21(5-6):323-330
180. Park J H, Lee J J, Yoon S, et al. Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle:identification of a novel muscle-preferred regulatory element [J]. Nucleic Acids Res,2001,29(14):3041-3050
181. Pegg J R, Balfry S K, Gordon L, et al. Stress, immune function and disease resistance in juvenile salmonids [J]. Bull Aquacult Assoc Can,1995,95(4):28-35
182. Pelham H R B. A regulatory upstream promoter element in the Drosophila Hsp70 heat-shock gene [J]. Cell,1982,30(2):517-528
183. Perez-Casanova J C, Rise M L, Dixon B, et al. The immune and stress responses of Atlantic cod to long-term increases in water temperature [J]. Fish Shellfish Immunol,2008,24(5):600-609
184. Peter M E, Heufelder A E, Hengartner M O. Advances in apoptosis research [J]. Proc Natl Acad Sci USA,1997,94(24):12736-12737
185. Peters G, Faisal M, Lang T, et al. Stress caused by social interaction and its effect on susceptibility to Aeromonas hydrophila infection in rainbow trout Salmo gairdneri [J]. Dis Aquat Org,1988,4: 83-89
186. Peters G, Nuβgen A, Raabe A, et al. Social stress induces structural and functional alterations of phagocytes in rainbow trout(Oncorhynchus mykiss) [J]. Fish Shellfish lmmunol,1991,1(1):17-31
187. Piano A, Asirelli C, Caselli F, et al. Hsp70 expression in thermally stressed Ostrea edulis, a commercially important oyster in Europe [J]. Cell Stress Chaperones,2002,7(3):250-257
188. Pickering A D. Environmental stress and the survival of brown trout, Salmo trutta [J]. Freshwater Biol,1989,21(1):47-55
189. Pickering A D. Introduction:The concept of biological stress [C]//Pickering A D. Stress and Fish. London:Academic Press,1981:1-9
190. Pottinger T G. Changes in blood cortisol, glucose and lactate in carp retained in anglers'keepnets [J]. J Fish Biol,1998,53(4):728-742
191. Price B D, Calderwood S K. Ca2+ is essential for multistep activation of the heat shock factor in permeabilized cells [J]. Mol Cell Biol,1991,11(6):3365-3368
192. Procarione L S, Barry T P, Malison J A. Effects of high rearing densities and loading rates on the growth and stress responses of juvenile rainbow trout [J]. N Am J Aquacult,1999,61(2):91-96
193. Ravagnan L, Gurbuxani S, Susin S A, et al. Heat-shock protein 70 antagonizes apoptosis-inducing factor [J]. Nat Cell Biol,2001,3(9):839-843
194. Reid S G, Bernier N J, Perry S F. The adrenergic stress response in fish:control of catecholamine storage and release [J]. Comp Biochem Physiol C,1998,120(1):1-27
195. Reed K H, Larsen H J S, Linder R D, et al. Genetic variation in lysozyme activity in rainbow trout (Oncorhynchus mykiss) [J]. Aquaculture,1993,109(3-4):237-244
196. Rojtinakem J, Hirono I, Itami T. Gene expression in haemocytes of Kuruma prawn, Penaeus japonicus, in response infection with WSSV by KST approach [J]. Fish Shellfish Immunol,2002, 13(1):69-83
197. Rose T M, Schultz E R, Henikoff J G, et al. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences [J]. Nucleic Acids Res,1998,26(7):1628-1635
198. Rotllant J, Pavlidis M, Kentouri M, et al. Non-specific immune responses in the red porgy Pagrus pagrus after crowding stress [J]. Aquaculture,1997,156(3-4):279-290
199. Rotllant J, Tort L. Cortisol and glucose responses after acute stress by net handling in the sparid red porgy previously subjected to crowding stress [J]. J Fish Biol,1997,51(1):21-28
200. Ruane N M, Komen H. Measuring cortisol in the water as an indicator of stress caused by increased loading density in common carp(Cyprinus carpio) [J]. Aquaculture,2003,218(1-4):685-693
201. Ruane N M, Nolan D T, Rotllant J, et al. Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Kr(?)yer) influences the physiological response to an acute stressor [J]. Fish Shellfish Immunol,2000,10(5): 451-463
202. Sadler J, Wells R M G, Pankhurst P M, et al. Blood oxygen transport, rheology and haematological responses to confinement stress in diploid and triploid Atlantic salmon, Salmo salar [J]. Aquaculture,2000,184(3-4):349-361
203. Sahin K, Sahin N, Kucuk O. Effects of chromium, and ascorbic acid supplementation on growth, carcass traits, serum metabolites, and antioxidant status of broiler chickens reared at a high ambient temperature (32℃) [J]. Nutr Res,2003,23(2):225-238
204. Sahu S, Das B K, Pradhan J, et al. Effect of Magifera indica kernel as a feed additive on immunity and resistance to Aeromonas hydrophila in Labeo rohita fingerlings [J]. Fish Shellfish Immunol, 2007,23(1):109-118
205. Sakai D K. The assessment of the health condition of salmonids by non-specific haemolytic (SH50) activity of serum [J]. Bull Jpn soc sci fish,1983,49(10):1487-1491
206. Sakai M. Current research status of fish immunostimulants [J]. Aquaculture,1999,172(1-2):63-92
207. Sakakura Y, Koshio S, Iida Y, et al. Dietary vitamin C improves the quality of yellowtail (Seriola quinqueradiata) seedlings [J]. Aquaculture,1998,161(1-4):427-436
208. Saleh A, Srinivasula S M, Balkir L, et al. Negative regulation of the Apaf-1 apoptosome by Hsp70 [J]. Nat Cell Biol,2000,2:476-483
209. Santoro M G. Heat shock factors and the control of the stress response [J] Biochem Pharmacol, 2000,59(1) 55-63
210. Scarim A L, Heitmeier M R, Corbett J A. Heat shock inhibits cytokine-induced nitric oxide synthase expression by rat and human islets [J]. Endocrinology,1998,139(12):5050-5057
211. Schreck C B, Contreras-Sanchez W, Fitzpatrick M S. Effects of stress on fish reproduction, gamete quality, and progeny [J]. Aquaculture,2001,197(1-4):3-24
212. Selvakumar S, Geraldine P. Heat shock protein induction in the freshwater prawn Macrobrachium malcolmsonii:acclimation-influenced variations in the induction temperatures for Hsp70 [J]. Comp Biochem Physiol A,2005,140(2):209-215
213. Selye H. Stress and the general adaptation syndrome [J]. Br Med J,1950,1:1383-1392
214. Shankaran M, Yamamoto B K, Gudelsky G A. Ascorbic acid prevents 3,4-methylenedioxy methamphetamine (MDMA)-induced hydroxyl radical formation and the behavioral and neurochemical consequences of the depletion of brain 5-HT [J]. Synapse,2001,40(1):55-64
215. Shiau S Y, Hsu T S. Vitamin C requirement of grass shrimp, Penaeus monodon, as determined with L-ascorbyl-2-monophosphate [J]. Aquaculture,1994,122(4):347-357
216. Shimeno S, Shikata T, Hosokawa H, et al. Metabolic response to feeding rates in common carp, Cyprinus carpio [J]. Aquaculture,1997,151(1-4):371-377
217. Sies H. Oxidative stress:oxidants and antioxidants [J]. Exp Physiol,1997,82(2):291-295
218. Sinha A K. Colorimetric assay of catalase [J]. Anal Biochem,1972,47(2):389-394
219. Sistonen L, Sarge K D, Phillips B, et al. Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells [J]. Mol Cell Biol,1992,12(9):4104-4111
220. Sobhana K S, Mohan C V, Shankar K M. Effect of dietary vitamin C on the disease susceptibility and inflammatory response of mrigal, Cirrhinus mrigala (Hamilton) to experimental infection of Aeromonas hydrophila [J]. Aquaculture,2002,207(3-4):225-238
221. Soliman A K, Jauncey K, Roberts R J. The effect of dietary ascorbic aicd supplementation on hatchability, survival rate and fry performance in Oreochromis mossambicus (Peters) [J]. Aquaculture,1986,59(3-4):197-208
222. Sommer C V, Binkowski F P, Schalk M A, et al. Stress factors that can affect studies of drug metabolism in fish [J]. Vet Hum Toxicol,1986,28(Suppl 1):45-54
223. Song L S, Wu L T, Ni D J, et al. The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacterial challenge and naphthalin stress [J]. Fish Shellfish Immunol,2006,21(4):335-345
224. S(?)rensen J G, Kristensen T N, Loeschcke V. The evolutionary and ecological role of heat shock proteins [J]. Ecol Lett,2003,6(11):1025-1037
225. Sorger P K, Lewis M J, Pelham H R. Heat shock factor is regulated differently in yeast and HeLa cells [J]. Nature,1987,329(6134):81-84
226. Sorger P K, Pelham H R. Purification and characterization of a heat-shock element binding protein from yeast [J]. EMBO J,1987,6(10):3035-3041
227. Sovenyi J, Szakolczai J. Studies on the toxic and immunosuppressive effects of cadmium on the common carp [J]. Acta Vet Hung,1993,41(3-4):415-426
228. Sreedhar A S, Csermely P. Heat shock proteins in the regulation of apoptosis:new strategies in tumor therapy A comprehensive review [J]. Pharmacol Ther,2004,101(3):227-257
229. Srinivas G, Anto R J, Srinivas P, et al. Emodin induces apoptosis of human cervical cancer cells through poly(ADP-ribose) polymerase cleavage and activation of caspase-9 [J]. Eur J Pharmacol, 2003,473(2-3):117-125
230. Steffens W. Principles of fish nutrition [M]. New York:Ellis Horwood Press,1989:256-258
231. Stephens S M, Alkindi A Y A, Waring C P et al. Corticosteroid and thyroid responses of larval and juvenile turbot exposed to the water-soluble fraction of crude oil [J]. J Fish Biol,1997,50(5): 953-964
232. Stuart R A, Cyr D M, Neupert W. Hsp70 in mitochondrial biogenesis:From chaperoning nascent polypeptide chains to facilitation of protein degradation [J]. Experientia,1994,50(11-12): 1002-1011
233. Sumpter J P, Dye H M, Benfey T J. The effects of stress on plasma ACTH, a-MSH, and cortisol levels in salmonid fishes [J]. Gen Comp Endocrinol,1986,62(3):377-385
234. Sun L T, Chen G R, Chang C F. Acute response of blood parameters and comatose effects in salt-acclimated tilapias exposed to low temperatures [J]. J Therm Biol,1995,20(3):299-306
235. Sun P M, Liu Y T, Zhao Y G, et al. Relationship between heart damages and HSPs mRNA in persistent heat stressed broilers [J]. Agric Sci China,2007,6(2):227-233
236. Sunyer J O, Gomez E, Tort L, et al. Physiological responses and depression of humoral components of the immune system in gilthead sea bream (Sparus aurata) following daily acute stress [J]. Can J Fish Aquat Sci,1995,52(11):2339-2346
237. Tamura Y, Peng P, Liu K, et al. Immunotheraphy of tumors with autologous tumor-derived heat shock protein preparations [J]. Science,1997,278(5335):117-120
238. Tedengren M, Olsson B, Reimer O, et al. Heat pretreatment increases cadmium resistance and HSP70 levels in Baltic Sea mussels [J]. Aquatic Toxicol,2000,48(1):1-12
239. Thompson I, White A, Fletcher T C, et al. The effect of stress on the immune response of Atlantic salmon (Salmo salar L.) fed diets containing different amounts of vitamin C [J]. Aquaculture,1993, 114(1-2):1-18
240. Van Raaij M T, Pit D S, Balm P H, et al. Behavioral strategy and the physiological stress response in rainbow trout exposed to severe hypoxia [J]. Horm Behav,1996,30(1):85-92
241. Van Weerd J H, Komen J. The effects of chronic stress on growth in fish:a critical appraisal [J]. Comp Biochem Physiol A,1998,120(1):107-112
242. Vasudeva Rao Y, Das B K, Jyotyrmayee P, et al. Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila [J]. Fish Shellfish Immunol,2006, 20(3):263-273
243. Verlhac V, Gabaudan J, Obach A, et al. Influence of dietary glucan and vitamin C on non-specific and specific immune responses of rainbow trout(Oncorhynchus mykiss) [J]. Aquaculture,1996, 143(2):123-133
244. Verlhac V, Gabaudan J. Influence of vitamin C on the immune system of salmonids [J]. Aquacult Res,1994,25(1):21-36
245. Vijayan M M, Leatherland J F. High stocking density affects cortisol secretion and tissue distribution in brook charr, Salvelinus fontinalis [J]. J Endocrinol,1990,124(2):311-318
246. Waagbe R, Glette J, Raa-Nilsen E, et al. Dietary vitamin C immunity and disease resistance in Atlantic salmon (Salmo salar) [J]. Fish Physiol Biochem,1993,12(1):61-73
247. Wahli T, Verlhac V, Girling P, et al. Influence of dietary vitamin C on the wound healing process in rainbow trout(Oncorhynchus mykiss) [J]. Aquaculture,2003,225(1-4):371-386
248. Wang H H, Chung J G. Emodin-induced inhibition of growth and DNA damage in the Helicobacter pylori [J]. Curr Microbiol,1997,35(5):262-266
249. Wang X J, Kim K W, Bai S C. Effects of different dietary levels of L-ascorbyl-2-polyphosphate on growth and tissue vitamin C concentrations in juvenile olive flounder, Paralichthys olivaceus (Temminck et Schlegel) [J]. Aquae Res,2002,33(4):261-267
250. Wang Y, Xiong L, Yang K J, et al. Effect of beta-cypermethrin on GPT and GOT activities of crucian serum [J]. Agric Sci Technol,2005,6(1):20-23
251. Welch W J. How cells respond to stress [J]. Sci Am,1993,268(5):56-64
252. Welch W J. Mammalian stress response:cell physiology, structure/function of stress proteins, and implications for medicine and disease [J]. Physiol Rev,1992,72(4):1063-1081
253. Westwood J T, Clos J, Wu C. Stress-induced oligomerization and chromosomal relocalization of heat-shock factor [J]. Nature,1991,353(6347):822-827
254. Wilson R P. Absence of ascorbic acid synthesis in channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus) [J]. Comp Biochem Physiol B,1973,46(3):635-638
255. Wu C, Zimarino C, Tsai B. Transcriptional regulation of heat shock genes [C]//Morimoto R I, Tissieres A, Georgopoulos C. Stress Proteins in Biology and Medicine. New York:Cold Spring Harbor Laboratory,1990:429-442
256. Wu Y, Wan T, Zhou X, et al. Hsp70-like protein 1 fusion protein enhances induction of carcinoembryonic antigen-specific CD8+CTL response by dendritic cell vaccine [J]. Cancer Res, 2005,65(11):4947-4954
257. Xiao C, Chen S, Yuan M, et al. Expression of the 60 kDa and 71 kDa heat shock proteins and presence of antibodies against the 71 kDa heat shock protein in pediatric patients with immune thrombocytopenic purpura [J]. BMC Blood Disord,2004,4(1):1-10
258. Xie J, Liu B, Zhou Q L, et al. Effects of anthraquinone extract from rhubarb Rheum officinale Baill on the crowding stress response and growth of common carp Cyprinus carpio var. Jian [J]. Aquaculture,2008,281(1-4):5-11
259. Yamashita M, Hirayoshi K, Nagata K. Characterization of multiple members of the HSP70 family in platyfish culture cells:molecular evolution of stress protein HSP70 in vertebrates [J]. Gene,2004, 336(2):207-218
260. Yin G J, Ardo L, Thompson K D, et al. Chinese herbs (Astragalus radix and Ganoderma lucidum) enhance immune response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila [J]. Fish Shellfish Immunol,2009,26(1):140-145
261. Yin G J, Jeney G, Racz T, et al. Effect of two chinese herbs(Astragalus radix and Scutellaria radix) on non-specific immune response of tilapia, Oreochromis niloticus [J]. Aquaculture,2006,253(1-4): 39-47
262. Yin Z, Lam T J, Sin Y M. The efects of crowding stress on the non-specific immune response in fancy carp(Cyprinus carpio L.) [J]. Fish Shellfish Immunol,1995,5(7):519-529
263. Yost H J, Lindquist S. RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis [J]. Cell,1986,45(2):185-193
264. Zamzami N, Kroemer G.The mitochondrion in apoptosis:How Pandora's box opens [J]. Nat Rev Mol Cell Biol,2001,2(1):67-71
265. Zarate J, Bradley T M. Heat shock proteins are not sensitive indicators of hatchery stress in salmon [J]. Aquaculture,2003,223(1-4):175-187
266. Zhu X T, Zhao X, Burkholder W F, et al. Structural analysis of substrate binding by the molecular chaperone DnaK [J]. Science,1996,272(5268):1606-1614
267.艾春香,陈立桥,周忠良,等.维生素C、E对中华绒螯蟹生殖性能的影响[J].水产学报,2003,27(1)：62-68
268.陈琦,华修国,顾晓峰,等.复方中草药对大鼠热应激蛋白70mRNA的表达影响[J].上海交通大学学报(农业科学版),2006,24(1)：61-64
269.陈孝煊,吴志新,殷居易,等.大黄、穿心莲、板蓝根和金银花对异育银鲫免疫机能的影响[J].中国水产科学,2003,10(1)：36-40
270.陈寅儿,金珊,王国良.鲈鱼溶藻弧菌病的血液生理生化指标研究[J].台湾海峡,2005,24(1)：104-108
271.辜玲芳,侯永清,丁斌鹰,等.几种植物提取物对异育银鲫生长性能和血液生化指标的影响[J].淡水渔业,2008,38(2)：23-26
272.郭松林,关瑞章,冯建军,等.嗜水气单胞菌感染对美洲鳗鲡血.液和生化指标的影响[J].集美大学学报(自然科学版),2010,15(2)：93-97
273.侯炜,杨占秋,陈科力,等.大黄有效成分抗单纯疱疹病毒作用的实验研究[J].药物分析杂志2003,23(4)：259-262
274.胡斌,李小勤,冷向军,等.饲料Vc对草鱼生长、肌肉品质及非特异性免疫的影响[J].中国水产科学,2008,15(5)：794-800
275.胡吕江,马烈,何学梅,等.九制大黄葸醌衍生物对动物高血脂及血液流变学的影响[J].中成药,2001,23(1)：31-33
276.黄泰康.常用中药成分与药理手册[M].北京：中国医药科技出版社,1994：228
277.吉红,曹福余,于海淼.黄岑素对鲤鱼健康及生长的影响[J].安徽农业大学学报,2008,35(4)：535-539
278.姜晓峰,甄永苏.大黄素逆转肿瘤细胞多药抗药性的作用[J].药学学报,1999,34(3)：164-167
279.金抗,于东云,由田.大黄对急性肺损伤大鼠热休克蛋白70表达的影响[J].中国急救医学,2007,27(9)：827-829
280.雷爱莹,曾地刚.复方中草药对凡纳滨对虾热应激蛋白70基因表达的影响[J].广西农业科学,2008,39(6)：830-833
281.李爱华.拥挤胁迫对草鱼皮质醇、血糖及肝脏中抗坏血酸含量的影响[J].水生生物学报,1997,21(4)：384-386
282.李爱杰.水产动物营养与饲料学[M].北京：中国农业出版社,1996：55
283.李桂峰,钱沛锋,孙际佳,等.维生素C对胡子鲶Claris fuscus细胞活性和血清因子的影响[J].中山大学学报(自然科学版),2005,44(5)：75-83
284.李桂峰,钱沛锋,孙际佳,等.维生素C对胡子鲶血清免疫相关酶活性的影响[J].大连水产学院学报,2004,19(4)：301-305
285.廖昌容,陈毕生.抗生素在水产动物体内的药代动力学研究进展[J].中国兽药杂志,2004,38(3)：30-34
286.刘红柏,黄江,马爱枝,等.中草药复方添加剂对施氏鲟生长及血液生化指标的影响[J].水产学杂志,2009,22(3)：14-18
287.刘红柏,张颖,卢彤岩,等.饲料中添加中草药对鲤鱼免疫功能的影响[J].集美大学学报(自然科学版),2004,9(4)：317-321
288.陆承平.致病性嗜水气单胞菌及其所致鱼病综述[J].水产学报,1992,16(3)：282-288
289.马得莹,单安山.几种中草药抗蛋鸡热应激作用分子机制的初步研究[J].动物营养学报,2007,19(3)：283-288
290.麦康森,艾庆辉,徐玮,等.水产养殖中的环境胁迫及其预防--营养学途径[J].中国海洋大学学报,2004,34(5)：767-774
291.明建华,谢骏,刘波,等.团头鲂HSP70 cDNA的克隆、序列分析以及热应激对其mRNA表达的影响[J].中国水产科学,2009,16(5)：635-648
292.聂品.鱼类非特异性免疫研究的新进展[J].水产学报,1997,21(1)：69-73
293.饶玉才,胡宝庆,文春根.嗜水气单胞菌感染对背角无齿蚌5种免疫相关酶活力的影响[J].水生生物学报,2009,33(3)：406-412
294.史洪博,段秀梅,李树蕾,等.结核分支杆菌HSP70原核表达载体的构建和表达及蛋白纯化[J].吉林大学学报(医学版),2006,32(6)：989-992,999
295.舒筱灿,周利玲,吴和平,等.大黄素对动脉粥样硬化的干预[J].中国组织工程研究与临床康复,2007,11(34)：6856-6859
296.苏洁,蔡宏,朱玉贤.结核分枝杆菌热休克蛋白Hsp70在原核表达载体中的克隆表达与鉴定[J].中国人兽共患病学报,2006,22(8)：712-715
297.孙峰,张煜,李立德,等.感染嗜水气单胞菌对鲫鱼非特异性免疫功能的影响[J].中国海洋大学学报,2005,35(5)：815-818
298.王洪琦,邹素芬,李建国.清热复方对热休克大鼠肝、肺组织热休克蛋白基因表达的调控作用[J].广州中医药大学学报,2004,21(1)：37-39
299.王镜岩,朱圣庚,徐长法.生物化学[M].3版.北京：高等教育出版社,2006：582-583
300.王群,丁银娣,赵云龙,等.维生素C对中华绒螯蟹雄性生殖的影响[J].动物学杂志,2004,39(2)：1-5
301.王文博,汪建国,李爱华,等.拥挤胁迫后鲫鱼血液皮质醇和溶菌酶水平的变化及对病原的敏感性[J].中国水产科学,2004,11(5)：408-412
302.王文俊,吴咸中,姚智,等.大黄素、丹参素对单核细胞分泌炎性细胞因子的调节[J].中国免疫学杂志,1995,11(6)：370-372
303.王小玲.应激对黄颡鱼非特异性免疫细胞的影响[D].武汉：华中农业大学,2006：34
304.席峰.鱼类应激及其适应性生热作用[J].饲料研究,2001,10：10-12
305.谢数涛,吴任,许忠能,等.亚致死温度热休克对凡纳滨对虾的保护作用[J].中山大学学报(自然科学版),2005,44(6)：87-89
306.谢一荣,吴锐全,谢骏,等.维生素C水平对大口黑鲈抵抗嗜水气单胞菌感染的影响[J].水利渔业,2007,27(5)：102-104
307.徐小芳,罗燕,赵民,等.中药复方多糖对鸡抗氧化功能的影响[J].中国农业科学,2009,42(2)：706-713
308.徐增光,杨进波,杨杪,等.抗氧化维生素对苯并(a)芘抑制内皮细胞HSP70表达的干预作用[J].环境与职业医学,2004,21(2)：111-113
309.姚泰.生理学[M].北京：人民卫生出版社,2002：367407
310.展玉涛,刘宾,李定国,等.大黄素抗肝纤维化的作用机制[J].中华肝脏病杂志,2004,12(4)：245-246
311.展玉涛,魏红山.大黄素对大鼠四氯化碳性肝损伤保护作用的实验研究[J].中国中医药科技,2000,7(1)：30-31
312.张骏,翁福海,李会强,等.大黄素对内毒素刺激下的大鼠腹腔巨噬细胞分泌TNFα及NO的影响[J].天津医科大学学报,2001,7(2)：189-191
313.张廷军,扬振才,孙儒泳.鱼类对高密度环境的适应[J].水产科技情报,1998,25(3)：110-113
314.周利玲,舒筱灿,吴和平,等.大黄素干预鹌鹑脂肪肝病变的效果[J].中国临床康复,2006,10(32)：57-59,F0003
315.周显青,李胜利,王晓辉,等.维生素C多聚磷酸酯对小鼠肝脏脂质过氧化物和抗氧化物酶的影响[J].动物学报,2004,50(3)：370-374
316.周显青,牛翠娟,孙儒泳.维生素C和酸应激对中华鳖幼鳖血清补体C3和C4含量的影响[J].动物学报,2003,49(6)：769-774
317.周显青,乔欣.维生素C多聚磷酸酯对小鼠抗热应激能力的影响[J].动物学研究,2004,25(5)：447451
318.周玉,郭文场,杨振国,等.欧洲鳗鲡“狂游病”血液生化指标研究[J].水生生物学报,2002,26(3)：314-316
319.周玉,郭文场,杨振国,等.鱼类血液指标的研究进展[J].上海水产大学学报,2001,10(2)：163-165
2. Affonso E G, Silva Eda C, Tavares-Dias M, et al. Effect of high levels of dietary vitamin C on the blood responses of matrinxa (Brycon amazonicus) [J]. Comp Biochem Physiol A,2007,147(2): 383-388
3. Ai Q H, Mai K S, Tan B P, et al. Effects of dietary vitamin C on survival, growth, and immunity of large yellow croaker, Pseudosciaena crocea [J]. Aquaculture,2006,261(1):327-336
4. Airaksinen S, Jokilehto T, Rabergh C M I, et al. Heat-and cold-inducible regulation of HSP70 expression in zebrafish ZF4 cells [J]. Comp Biochem Physiol B,2003,136(2):275-282
5. Ali K S, Dorgai L, Abraham M, et al. Tissue-and stressor-specific differential expression of two hsc70 genes in carp [J]. Biochem Biophys Res Commun,2003,307(3):503-509
6. Alves D S, Perez-Fons L, Estepa A, et al. Membrane-related effects underlying the biological activity of the anthraquinones emodin and barbaloin [J]. Biochem Pharmacol,2004,68(3):549-561
7. Amin J, Fernandez M, Ananthan J, et al. Cooperative binding of heat shock transcription factor to the Hsp70 promoter in vivo and in vitro [J]. Biol Chem,1994,269(7):4804-4811
8. Anderson D P, Dixon O W. Suppression of antibody-producing cells in rainbow trout spleen sections exposed to copper in vitro [J]. J Aquat Anim Health,1989,1(1):57-61
9. Anderson D P. Immunostimulants, adjuvants and vaccine carrier in fish:application to aquaculture [J]. Annu Rev Fish Dis,1992,2:281-307
10. Anderson I, Brass A. Searching DNA databases for similarities to DNA sequences:when is a match significant? [J] Bioinformatics,1998,14(4):349-356
11. Angelidis C E, Lazaridis I, Pagoulatos G N. Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance [J]. Eur J Biochem,1991,199(1):35-39
12. Ansary A, Haneef R M, Torres J L, et al. Plasmids and antibiotic resistance in Aeromonas hydrophila isolated in Malaysia from healthy and diseased fish [J]. J Fish Dis,1992,15(2):191-196
13. Arnold K, Bordoli L, Kopp J, et al. The SWISS-MODEL workspace:a web-based environment for protein structure homology modeling [J]. Bioinformatics,2006,22(2):195-201
14. Baler R, Dahl G, Voellmy R. Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1 [J]. Mol Cell Biol,1993,13(4):2486-2496
15. Barnett C W, Pankhurst N W. The effects of common laboratory and husbandry practices on the stress response of greenback flounder Rhombosoleu tapirina (Giinther,1862) [J]. Aquaculture, 1998,162(3-4):313-329
16. Barton B A, Iwama G K. Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids [J]. Annual Rev Fish Dis,1991,1:3-26
17. Basu N, Todgham A E, Ackerman P A, et al. Heat shock protein genes and their functional significance in fish [J]. Gene,2002,295(2):173-183
18. Bayne C J, Gerwick L. The acute phase response and innate immunity of fish [J]. Dev Comp Immunol,2001,25(8-9):725-743
19. Beere H M, Wolf B B, Cain K, et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome [J]. Nat Cell Biol,2000,2(8):469-475
20. Bernelli-Zazzera A, Cairo G, Schiaffonati L, et al. Stress proteins and reperfusion stress in the liver [J]. Ann N Y Acad of Sci,1992,663:120-124
21. Bernier N J, Peter R E. The hypothalamic-pituitary-interrenal axis and the control of food intake in teleost fish [J]. Comp Biochem and Physiol B,2001,129(2-3):639-644
22. Blom J H, Dabrowski K. Ascorbic acid metabolism in fish:is there a maternal effect on the progeny? [J]. Aquaculture,1996,147(3-4):215-224
23. Boone A N, Vijayan M M. Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes:effect of heat shock and heavy metal exposure [J]. Comp Biochem Physiol C,2002, 132(2):223-233
24. Bork P, Sander C, Valencia A. An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins [J]. Proc Natl Acad Sci USA,1992,89(16): 7290-7294
25. Boutet I, Tanguy A, Rousseau S, et al. Molecualr identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas [J]. Cell Stress Chaperones,2003,8 (1):76-85
26. Breathnach R, Chambon P. Organization and expression of eucaryotic split genes coding for proteins [J]. Annu Rev Biochem,1981,50:349-383
27. Brock P, Eldred E W, Woiszwillo J E, et al. Direct solid-phase 1251 radioimmunoassy of serum cortisol [J]. Clin Chem,1978,24(9):1595-1598
28. Brown S B, MacLatchy D L, Hara T J, et al. Effects of ambient pH and aluminum on plasma kinetics of cortisol, T3, and T4 in rainbow trout(Oncorhynchus mykiss) [J]. Can J Zool,1990,68(7): 1537-1543
29. Burdon R H. Heat shock and the heat shock proteins [J]. Biochem J,1986,240(2):313-324
30. Burdon R H. Heat shock proteins in relation to medicine [J]. Mol Aspects Med,1993,14(2):83-165
31. Cao Y, Ohwatari N, Matsumoto T, et al. TGF-β1 mediates 70-kDa heat shock protein induction due to ultraviolet irradiation in human skin fibroblasts [J]. Pflugers Arch,1999,438(3):239-244
32. Cara J B, Aluru N, Moyano F J, et al. Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout [J]. Comp Biochem Physiol,2005,142(4): 426-431
33. Castelli C, Ciupitu A M, Rini F, et al. Human heat shock protein 70 peptide complexes specifically activate antimelanoma T cells [J]. Cancer Res,2001,61(1):222-227
34. Chang C H, Lin C C, Yang J J, et al. Anti-inflammatory effects of emodin from ventilago leiocarpa [J]. Am J Chin Med,1996,24(2):139-142
35. Chen R, Lochmann R, Goodwin A, et al. Effects of dietary vitamins C and E on alternative complement activity, hematology, tissue composition, vitamin concentrations and response to heat stress in juvenile golden shiner (Notemigonus crysoleucas) [J]. Aquaculture,2004,242(1-4): 553-569
36. Chen W H, Sun L T, Tsai C L, et al. Cold-stress induced the modulation of catecholamines, cortisol, immunoglobulin M, and leukocyte phagocytosis in tilapia [J]. Gen Comp Endocrinol,2002, 126(1):90-100
37. Cheng P Z, Liu X, Zhang G F, et al. Cloning and expression analysis of a HSP70 gene from pacific abalone (Haliotis discus hannai) [J]. Fish Shellfish Immunol,2007,22(1-2):77-87
38. Chilmonczyk S. Rainbow trout lymphoid organs:cellular effects of corticosteroids and anti-thymocyte serum [J]. Dev Comp Immunol,1982,6(2):271-280
39. Clearwater S J, Pankhurst N W. The response to capture and confinement stress of plasma cortisol, plasma sex steroids and vitellogenic oocytes in the marine teleost, red gurnard [J]. J Fish Biol,1997, 50(2):429-441
40. Clerton P, Troutaud D, Verlhac V, et al. Dietary vitamin E and rainbow trout (Oncorhynchus my kiss) phagocyte functions:effect on gut and on head kidney leucocytes [J]. Fish Shellfish Immunol,2001, 11(1):1-13
41. Collazos M E, Barriga C, Ortega E. Effect of high summer temperatures upon granulocyte phagocytic function of the tench (Tinea tinea, L.) [J]. Comp Immunol Microbiol infect Dis,1995, 18(2):115-121
42. Creagh E M, Carmody R J, Cotter T G. Heat shock protein 70 inhibits caspase-dependent and-independent apoptosis in Jurkat T cells [J]. Exp Cell Res,2000,257(1):58-66
43. Dabrowska H, Dabrowski K, Meyer-Burgdorff K, et al. The effect of large doses of vitamin C and magnesium on stress responses in common carp, Cyprinus carpio [J]. Comp Biochem Physiol A, 1991,99(4):681-685
44. Dabrowski K, Ciereszko A. Ascorbic acid protects against male infertility in a teleost fish [J]. Experientia,1996,52(2):97-100
45. Dang S S, Zhang Z G, Chen Y R, et al. Inhibition of the replication of hepatitis B virus in vitro by emodin [J]. Med Sci Monit,2006,12(9):302-306
46. Das D K, Engelman R M, Kimura Y. Molecular adaptation of cellular defences following preconditioning of the heart by repeated ischaemia [J]. Cardiovasc Res,1993,27(4):578-584
47. Das P, Gupta A, Manna S K. Heat shock protein 70 expression in different tissues of Cirrhinus mrigala (Ham.) following heat stress [J]. Aquae Res,2005,36(6):525-529
48. Dave G, Johansson-Sjobeck M L, Larsson A, et al. Effects of cortisol on the fatty acid composition of the total blood plasma lipids in the European eel, Anguilla anguilla L. [J]. Comp Biochem Physiol A,1979,64(1):37-40
49. de Menezes G C, Tavares-Dias M, Ono E A, et al. The influence of dietary vitamin C and E supplementation on the physiological response of pirarucu, Arapaima gigas, in net culture [J]. Comp Biochem Physiol A,2006,145(2):274-279
50. Deane E E, Li J, Woo N Y. Modulated heat shock protein expression during pathogenic Vibrio alginolyticus stress of sea bream [J]. Dis Aquat Organ,2004,62(3):205-215
51. Deane E E, Woo N Y S. Cloning and characterization of the hsp70 multigene family from silver sea bream:Modulated gene expression between warm and cold temperature acclimation [J]. Biochem Biophys Res Commun,2005,330(3):776-783
52. Demand J, Liiders J, Hohfeld J. The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors [J]. Mol Cell Biol,1998,18(4):2023-2028
53. Demers N E, Bayne C J. The immediate effects of stress on hormones and plasma lysozyme in rainbow trout [J]. Dev Comp Immunol,1997,21(4):363-373
54. Di Marco P, Priori A, Finoia M G, et al. Physiological responses of European sea bass Dicentrarchus labrax to different stocking densities and acute stress challenge [J]. Aquaculture, 2008,275(1-4):319-328
55. DuBeau S F, Pan F, Tremblay G C, et al. Thermal shock of salmon in vivo induces the heat shock protein hsp70 and confers protection against osmotic shock [J]. Aquaculture,1998,168(1-4): 311-323
56. Dziewulska-Szwajkowska D, Lozinska-Gabska M, Adamowicz A, et al. The effect of high dose of cortisol on glucose-6-phosphatase and fructose-1,6-bisphosphatase activity, and glucose and fructose-2,6-bisphosphate concentration in carp tissues (Cyprinus carpio L.) [J]. Comp Biochem Physiol B,2003,135(3):485-491
57. Ellis R J. The molecular chaperone concept [J]. Semin Cell Biol,1990,1(1):1-9
58. Ellsaesser C F, Clem L W. Haematological and immunological changes in channel catfish stressed by handling and transport [J]. J Fish Biol,1986,28(4):511-521
59. Espelid S, Lekken G B, Steiro K, et al. Effects of cortisol and stress on the immune system in Atlantic salmon (Salmo salar L.) [J]. Fish Shellfish Immunol,1996,6(2):95-110
60. Feder M E, Hofmann G E. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology [J]. Annu Rev Physiol,1999,61(1):243-282
61. Fevolden S E, Refstie T, Rφed K. H. Selection for high and low cortisol stress response in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) [J]. Aquaculture,1991,95(1-2): 53-65
62. Fevolden S E, Rφed K H, Fjalestad K. T, et al. Poststress levels of lysozyme and cortisol in adult rainbow trout:heritabilities and genetic correlations [J]. J Fish Biol,1999,54(4):900-910
63. Fevolden S E, Rφed K H, Fjalestad K. A combined salt and confinement stress enhances mortality in rainbow trout (Oncorhynchus mykiss) selected for high stress responsiveness [J]. Aquaculture, 2003,216(1-4):67-76
64. Fevolden S E, Reed K H. Cortisol and immune characteristics in rainbow trout (Oncorhynchus mykiss) selected for high or low tolerance to stress [J]. J Fish Biol,1993,43(6):919-930
65. Fevolden S E, Rφed K H, Fjalestad K. A combined salt and confinement stress enhances mortality in rainbow trout (Oncorhynchus mykiss) selected for high stress responsiveness [J]. Aquaculture, 2003,216(1-4):67-76
66. Foo J T W, Lam T J. Retardation of ovarian growth and depression of serum steroid levels in the tilapia, Oreochromis mossambicus, by cortisol implantation [J]. Aquaculture,1993,115(1-2): 133-143
67. Fracalossi D M, Allen M E, Nichols D K, et al. Oscars, Astronotus ocellatus, have a dietary requirement for vitamin C [J]. J Nutr,1998,128(10):1745-1751
68. Fracalossi D M, Allen M E, Yuyama L K, et al. Ascorbic acid biosynthesis in Amazonian fishes [J]. Aquaculture,2001,192(2-4):321-332
69. Franzellitti S, Fabbri E. Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors [J]. Biochem Biophys Res Commun,2005,336(4):1157-1163
70. Freeman B A, Crapo J D. Biology of disease:free radicals and tissue injury [J]. Lab Invest,1982, 47(5):412-426
71. Freeman B C, Myers M P, Schumacher R, et al. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1 [J]. EMBO J,1995,14(10): 2281-2292
72. Gabai V L, Meriin A B, Mosser D D, et al. Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance [J]. J Biol Chem,1997,272(29):18033-18037
73. Garrido C, Gurbuxani S, Ravagnan L, et al. Heat shock proteins:endogenous modulators of apoptotic cell death [J]. Biochem Biophys Res Commun,2001,286(3):433-442
74. Goel R K, Das Gupta G, Ram S N, et al. Antiulcerogenic and anti-inflammatory effects of emodin, isolated from Rhamnus triquerta wall [J]. Indian J Exp Biol,1991,29(3):230-232
75. Gornati R, Papis E, Rimoldi S, et al. Rearing density influences the expression of stress-related genes in sea bass (Dicentrarchus labrax, L.)[J]. Gene,2004,341:111-118
76. Granelli K, Bjorck L, Appelqvist L A. The variation of superoxide dismutase (SOD) and xanthine oxidase (XO) activities in milk using an improved method to quantitate SOD activity [J]. J Sci Food Agric,1995,67(1):85-91
77. Grutter A S, Pankhurst N W. The effects of capture, handling, confinement and ectoparasite load on plasma levels of cortisol, glucose and lactate in the coral reef fish Hemigymnus melapterus [J]. J Fish Biol,2000,57(2):391-401
78. Gursu M F, Onderci M, Gulcu F, et al. Effects of vitamin C and folic acid supplementation on serum paraoxonase activity and metabolites induced by heat stress in vivo [J]. Nutr Res,2004, 24(2):157-164
79. Guy C L, Li Q B. The organization and evolution of the spinach stress 70 molecular chaperone gene family [J]. Plant Cell,1998,10(4):539-556
80. Haddy J A, Pankhurst N W. Stress-induced changes in concentrations of plasma sex steroids in black bream [J]. J Fish Biol,1999,55(6):1304-1316
81. Hall T J. Role of hsp70 in cytokine production [J]. Cell Mol Life Sci,1994,50(11-12):1048-1053
82. Hardie L J, Fletcher T C, Secombes C J. The effect of dietary vitamin C on the immune response of the Atlantic salmon (Salmo solar L.) [J]. Aquaculture,1991,95(3-4):201-214
83. Harrison C J, Bohm A A, Nelson H C. Crystal structure of the DNA binding domain of the heat shock transcription factor [J]. Science,1994,263(5144):224-227
84. Hartl F U, Hayer-Hartl M. Molecular chaperones in the cytosol:from nascent chain to folded protein [J]. Science,2002,295(5561):1852-1858
85. Hartl F U. Molecular chaperones in cellular protein folding [J]. Nature,1996,381(6583):571-579
86. Haug T, Kjuul A K, Stensvag K, et al. Antibacterial activity in four marine crustacean decapods [J].Fish Shellfish Immunol,2002,12(5):371-385
87. Helenius A, Aebi M. Intracellular functions of N-linked glycans [J]. Science,2001,291(5512): 2364-2369
88. Hendrick J P, Hartl F U. Molecular chaperone functions of heat-shock proteins [J]. Annu Rev Biochem,1993,62:349-384
89. Henrique M M F, Gomes E F, Gouillou-Coustans M F, et al. Infulence of supplementation of practical diets with vitamin C on growth and response to hypoxic stress of seabream, Sparus aurata [J]. Aquaculture,1998,161(1-4):415-426
90. Hightower L E. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides [J]. J Cell Physiol,1980,102(3):407-427
91. Hightower L E. Heat shock, stress proteins, chaperones, and proteotoxicity [J]. Cell,1991,66(2): 191-197
92. Hofmann G E, Buckley B A, Airaksinen S, et al. Heat-shock protein expression is absent in the Antarctic fish Trematomus bernacchii (family Nototheniidae) [J]. J Exp Biol,2000,203:2331-2339
93. Hrubec T C, Robertson J L, Smith S A, et al. The effect of temperature and water quality on antibody response to Aeromonas salmonicida in sunshine bass (Morone chrysops× Morone saxatilis) [J]. Vet Immunol Immunopathol,1996,50(1-2):157-166
94. Hsieh S L, Chen Y N, Kuo C M. Physiological responses, desaturase activity, and fatty acid composition in milkfish (Chanos chanos) under cold acclimation [J]. Aquaculture,2003,220(1-4): 903-918
95. Huang S S, Yeh S F, Hong C Y. Effect of anthraquinone derivatives on lipid peroxidation in rat heart mitochondria:structure activity relationship [J]. J Nat Prod,1995,58(9):1365-1371
96. Huang Y, Wimler K M, Carmichael G G. Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing [J]. EMBO J,1999,18(6):1642-1652
97. Hwang D F, Lin T K. Effect of temperature on dietary vitamin C requirement and lipid in common carp [J]. Comp Biochem Physiol B,2002,131(1):1-7
98. Iguchi K, Ogawa K, Nagae M, et al. The influence of rearing density on stress response and disease susceptibility of ayu (Plecoglossus altivelis) [J]. Aquaculture,2003,220(1-4):515-523
99. Ishibashi Y, Kato K, Ikeda S, et al. Effects of dietary ascorbic acid on tolerance to intermittent hypoxic stress in Japanese parrot fish [J]. Nippon Suisan Gakkaishi,1992,58(11):2147-2152
100. Ishioka H. Live Fish Transportation, Series of Fisheries Book 39 [M]. Tokyo:Kosesha Kosekaku, 1982:52-69
101. Israeli D, Kimmel E. Monitoring the behavior of hypoxia-stressed Carassius auratus using computer vision [J]. Aquacult Eng,1996,15(6):423-440
102. Israeli-Weinstein D, Kimmel E. Behavioral response of carp (Cyprinus carpio) to ammonia stress [J]. Aquaculture,1998,165(1-2):81-93
103. Ito A, Fujioka M, Tanaka K, et al. Screening of cytokines to enhance vaccine effects of heat shock protein 70-rich tumor cell lysate [J]. J Biosci Bioeng,2005,100(1):36-42
104. Iwama G K, Vijayan M M, Forsyth R B, et al. Heat shock proteins and physiological stress in fish [J]. Amer Zool,1999,39(6):901-909
105. Janssens P A, Waterman J. Hormonal regulation of gluconeogenesis and glycogenolysis in carp (Cyprinus carpio) liver pieces cultured in vitro [J]. Comp Biochem Physiol A,1988,91(3):451-455
106. Jayasuriya H, Koonchanok N M, Geahlen R L, et al. Emodin, a protein tyrosine kinase inhibitor from Polygonum cuspidatum [J]. J Nat Prod,1992,55(5):696-698
107. Jeney G, Galeotti M, Volpatti D, et al. Prevention of stress in rainbow trout(Oncorhynchus mykiss) fed diets containing different doses of glucan [J]. Aquaculture,1997,154(1):1-15
108. Jiao C Z, Wang Z Z, Li F H, et al. Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis [J]. Chin Sci Bull,2004, 49(22):2385-2393
109. Jing X, Ueki N, Cheng J, et al. Induction of apoptosis in hepatocellular carcinoma cell lines by emodin [J]. Jpn J Cancer Res,2002,93(8):874-882
110. Johnston R N, Kucey B L. Competitive inhibition of hsp70 gene expression causes thermosensitivity [J]. Science,1988,242(4885):1551-1554
111. Johnston W L, MacDonald E, Hilton J W. Relationships between dietary ascorbic acid status and deficiency, weight gain and brain neurotransmitter levels in juvenile rainbow trout, Salmo gairdneri [J]. Fish Physiol Biochem,1989,6(6):353-365
112. Kanazawa A. Effects of docosahexaenoic acid and phospholipids on stress tolerance of fish [J]. Aqauculture,1997,155(1-4):129-134
113. Khassaf M, McArdle A, Esanu C, et al. Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle [J]. J Physiol,2003,549(2):645-652
114. Kiang J G, Tsokos G C. Heat shock protein 70 kDa:molecular biology, biochemistry and physiology [J]. Pharmacol Ther,1998,80(2):183-201
115. Kitabchi A E. Ascorbic acid in steroidogenesis [J]. Nature,1967,215:1385-1386
116. Knowlton A A, Salfity M. Nuclear localization and the heat shock proteins [J]. J Biosci,1996, 21(2):123-132
117. Kohler M, Haller H, Hartmann E. Nuclear protein transport pathways [J]. Exp Nephrol,1999,7(4): 290-294
118. Kothary R K, Jones D, Candido E P M.70-Kilodalton heat shock polypeptides from rainbow trout: characterization of cDNA sequences [J]. Mol Cell Biol,1984,4(9):1785-1791
119. Koven W, Barr Y, Lutzky S, et al. The effect of dietary arachidonic acid (20:4n-6) on growth, survival and resistance to handling stress in gilthead seabream (Sparus aurata) larvae [J]. Aquaculture,2001,193(1-2):107-122
120. Kregel K C. Heat shock proteins:modifying factors in physiological stress responses and acquired thermotolerance [J]. J Appl Physiol,2002,92(5):2177-2186
121. Kroeger P E, Sarge K D, Morimoto R I. Mouse heat shock transcription factors 1 and 2 prefer a trimeric binding site but interact differently with the HSP70 heat shock element [J]. Mol and Cell Biol,1993,13(6):3370-3383
122. Kroemer G, Reed J C. Mitochondrial control of cell death [J]. Nat Med,2000,6(5):513-519
123. Kuhn E R, Geris K L, van der Geyten S, et al. Inhibition and activation of the thyroidal axis by the adrenal axis in vertebrates [J]. Comp Biochem Physiol A,1998,120(1):169-174
124. Kumari J, Sahoo P K. High dietary vitamin C affects growth, non-specific immune responses and disease resistance in Asian catfish, Clarias batrachus [J]. Mol Cell Biochem,2005,280(1-2):25-33
125. Landolt M L. The relationship betwen diet and the immune response of fish [J]. Aquaculture,1989, 79(1-4):193-206
126. Le Morvan C, Deschaux P, Troutaud D. Effects and mechanisms of environmental temperature on carp (Cyprinus carpio) anti-DNP antibody response and non-specific cytotoxic cell activity:a kinetic study [J]. Dev Comp Immunol,1996,20(5):331-340
127. Lee H Z. Protein kinase C involvement in aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell [J]. Br J Pharmacol,2001,134(5):1093-1103
128. Lewis J, Devin A, Miller A, et al. Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-κB activation [J]. J Biol Chem,2000,275(14):10519-10526
129. Li M H, Wise D J, Robinson E H. Effect of dietary vitamin C on weight gain, tissue ascorbate concentration, stress response, and disease resistance of Channel catfish Ictalurus punctatus [J]. J World Aquacult Soc,1998,29(1):1-8
130. Lim C, Lovell R T. Pathology of the vitamin C deficiency syndrome in channel catfish (Ictalurus punctatus) [J]. J Nutr,1978,108(7):1137-1146
131. Lim L C, Dhert P, Chew W Y, et al. Enhancement of stress resistance of the Guppy Poecilia reticulata through feeding with vitamin C supplement [J]. J World Aquacul Soc,2002,33(1):32-40
132. Lin C C, Chang C H, Yang J J, et al. Hepatoprotective effects of emodin from Ventilago Leiocarpa [J].J Ethnopharmacol,1996,52(2):107-111
133. Lin M F, Shiau S Y. Dietary L-ascorbic acid affects growth, nonspecific immune responses and disease resistance in juvenile grouper, Epinephelus malabaricus [J]. Aquaculture,2005,244(1-4): 215-221
134. Lindguist S, Craig E A. The heat-shock proteins [J]. Annu Rev Genet,1988,22:631-677
135. Lis J, Wu C. Heat shock factor [C]//. McKnight S L, Yamamoto K R. Transcriptional Regulation. New York:Cold Spring Harbor Laboratory,1992:907-930
136. Liu B, Xie J, Ge X P, et al. Effects of anthraquinone extract from Rheum officinale Baill on the growth performance and physiological responses of Macrobrachium rosenbergii under high temperature stress [J]. Fish Shellfish Immunol,2010,29(1):49-57
137. Liu J, Yang W J, Zhu X J, et al. Molecular cloning and expression of two HSP70 genes in the prawn, Macrobrachi um rosenbergii [J]. Cell Stress Chaperones,2004,9(3):313-323
138. Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method [J]. Methods,2001,25(4):402-408
139. Lowry O H, Rosebrough N J, Farr A L, et al. Protein measurement with the folin phenol reagent [J]. J Biol Chem,1951,193(1):265-275
140. Macconnell E, Barrows F T. Pathological changes associated with vitamin C deficiency in walleyes [J]. J Aquat Anim Health,1993,5(4):287-293
141.Mackett D B, Tam W H, Fryer J N. Histological changes in insulin-immunoreactive pancreatic β-cell, and suppression of insulin secretion and somatotrope activity in brook trout(Salvelinus fontinalis) maintained on reduced food intake or exposed to acidic environment [J]. Fish Physiol Biochem,1992,10(3):229-243
142. Mahmoud K Z, Edens F W, Eisen E J, et al. Ascorbic acid decreases heat shock protein 70 and plasma corticosterone response in broilers(Gallus gallus domesticus) subjected to cyclic heat stress [J]. Comp Biochem Physiol B,2004,137(1):35-42
143. Manojlovic N T, Solujic S, Sukdolak S, et al. Antifungal activity of Rubia tinctorum, Rhamnus frangula and Caloplaca cerina [J]. Fitoterapia,2005,76(2):244-246
144. Mazik P M, Tomasso J R, Brandt T M. Efects of dietary vitamin C on growth, caudal fin development, and tolerance of aquaculture-related stressors in channel catfish [J]. Prog Fish-Cult, 1987,49(1):13-16
145. Mazur C F, Iwama G K. Handling and crowding stress reduces the number of plaque-forming cells in Atlantic salmon [J]. J Aquat Anim Health,1993,5(2):98-101
146. Mbwambo Z H, Apers S, Moshi M J, et al. Anthranoid compounds with antiprotozoal activity from Vismia orientalis [J]. Planta Med,2004,70(8):706-710
147. McCormick S D, Shrimptom J M, Carey J B, et al. Repeated acute stress reduces growth rate of Atlantic salmon parr and alters plasma levels of growth hormone, insulin-like growth factor I and cortisol [J]. Aquaculture,1998,168(1-4):221-235
148. McCully J D, Myrmel T, Lotz M M, et al. The rapid expression of myocardial hsp 70 mRNA and the heat shock 70 kDa protein can be achieved after only a brief period of retrograde hyperthermic perfusion [J]. J Mol Cell Cardiol,1995,27(3):873-882
149. McGarry T J, Lindquist S. The preferential translation of Drosophila hsp70 mRNA requires sequences in the untranslated leader [J]. Cell,1985,42(3):903-911
150. McRae M P. Vitamin C supplementation lowers serum low-density lipoprotein cholesterol and triglycerides:a meta-analysis of 13 randomized controlled trials [J].J Chiropr Med,2008,7(2): 48-58
151.Mehlen P, Kretz-Remy C, Preville X, et al. Human hsp27, Drosophila hsp27 and human aB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFa-induced cell death [J]. EMBO J,1996,15(11):2695-2706
152. Meng K W, Lv Y, Yu L, et al. Effects of emodin and double blood supplies on liver regeneration of reduced size graft liver in rat model [J]. World J Gastroenterol,2005,11(19):2941-2944
153. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test [J]. Anal Biochem,1978,86(1):271-278
154. Milarski K L, Morimoto R I. Expression of human HSP70 during the synthetic phase of the cell cycle [J]. Proc Natl Acad Sci USA,1986,83(24):9517-9521
155. Ming J H, Xie J, Xu P, et al. Molecular cloning and expression of two HSP70 genes in the Wuchang bream (Megalobrama amblycephala Yih) [J]. Fish Shellfish Immunol,2010,28(3): 407-418
156. Mock A, Peters G. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution [J]. J Fish Biol,1990,37(6):873-885
157. Molina A, Biemar F, Muller F, et al. Cloning and expression analysis of an inducible HSP70 gene from tilapia fish [J]. FEBS Lett,2000,474(1):5-10
158. Mommsen T P, Vijayan M M, Moon T W. Cortisol in teleosts:dynamics, mechanisms of action, and metabolic regulation [J]. Rev Fish Biol Fisher,1999,9(3):211-268
159. Montero D, Marrero M, Izquierdo M S, et al. Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress [J]. Aquaculture,1999,171(3-4):269-278
160. Montero D, Tort L, Robaina L, et al. Low vitamin E in diet reduces stress resistance of gilthead seabream (Sparus aurata) juveniles [J]. Fish Shellfish Immunol,2001,11(6):473-490
161. Morimoto R I, Kroeger P E, Cotto J J. The transcriptional regulation of heat shock genes:a plethora of heat shock factors and regulatory conditions [J]. EXS,1996,77:139-163
162. Morimoto R I. Cells in stress:transcriptional activation of heat shock genes [J]. Science,1993, 259(5100):1409-1410
163. Mosser D D, Caron A W, Bourget L, et al. The chaperone function of hsp70 is required for protection against stress-induced apoptosis [J]. Mol Cell Biol,2000,20(19):7146-7159
164. Nakai A, Tanabe M, Kawazoe Y, et al. HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator [J]. Mol Cell Biol,1997,17(1):469-481
165. Navarre O, Halver J E. Disease resistance and humoral antibody production in rainbow trout fed high levels of vitamin C [J]. Aquaculture,1989,79(1-4):207-221
166. Ndong D, Chen Y Y, Lin Y H, et al. The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures [J]. Fish Shellfish Immunol,2007,22(6):686-694
167. Ng T B, Liu F, Lu Y, et al. Antioxidant activity of compounds from the medicinal herb Aster tataricus [J]. Comp Biochem Physiol C,2003,136(2):109-115
168. Niedzwiecki A, Reveillaud I, Fleming J E. Changes in superoxide dismutase and catalase in aging heat-shocked Drosophila [J]. Free Radic Res,1992,17(6):355-367
169. Ohta T. Further examples of evolution by gene duplication revealed through DNA sequence comparisons [J]. Genetics,1994,138(4):1331-1337
170. Ojolick E J, Cusack R, Benfey T J, et al. Survival and growth of all-female diploid and triploid rainbow trout (Oncorhynchus mykiss) reared at chronic high temperature [J]. Aquaculture,1995, 131(3-4):177-187
171. Olsvik P A, Lie K K, Jordal A E, et al. Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon [J]. BMC Mol Biol,2005,6:21
172. Ortuno J, Esteban M A, Meseguer J. Effect of high dietary intake of vitamin C on non-specific immune response of gilthead seabream (Sparus aurata L.) [J]. Fish Shellfish Immunol,1999,9(5): 429-443
173. Ortuno J, Esteban M A, Meseguer J. Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L.) innate immune response [J]. Fish Shellfish Immunol,2001,11(2):187-197
174. Ozrurk-Urek R, Bozkaya L A, Tarhan L. The effects of some antioxidant vitamin- and trace element-supplemented diets on activities of SOD, CAT, GSH-Px and LPO levels in chicken tissues [J]. Cell Biochem Funct,2001,19(2):125-132
175. Pankhurst N W, Van Der Kraak G, Peter R E. Evidence that the inhibitory effects of stress on reproduction in teleost fish are not mediated by the action of cortisol on ovarian steroidogenesis [J]. Gen Comp Endocrinol,1995,99(3):249-257
176. Pankhurst N W, Van Der Kraak G. Effects of stress on reproduction and growth of fish [C]//Iwama G K, Pickering A D, Sumpter J P, et al. Fish Stres and Health in Aquaculture. Society for Experimental Biology Seminar Series 62. Cambrige, UK:Cambrige Univ Pres,1997:73-93
177. Pankhurst N W, Van Der Kraak G. Evidence that acute stress inhibits ovarian steroidogenesis in rainbow trout in vivo, through the action of cortisol [J]. Gen Comp Endocrinol,2000,117(2): 225-237
178. Paolucci M, Esposito V, di Fiore M M, et al. Effects of short postcapture confinement on plasma reproductive hormone and corticosterone profiles in Rana esculenta during the sexual cycle [J]. Boll Zool,1990,57(3):253-259
179. Parihar M S, Dubey A K, Javeri T, et al. Changes in lipid peroxidation, superoxide dismutase activity, ascorbic acid and phospholipid content in liver of freshwater catfish Heteropneustes fossilis exposed to elevated temperature [J]. J Therm Biol,1996,21(5-6):323-330
180. Park J H, Lee J J, Yoon S, et al. Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle:identification of a novel muscle-preferred regulatory element [J]. Nucleic Acids Res,2001,29(14):3041-3050
181. Pegg J R, Balfry S K, Gordon L, et al. Stress, immune function and disease resistance in juvenile salmonids [J]. Bull Aquacult Assoc Can,1995,95(4):28-35
182. Pelham H R B. A regulatory upstream promoter element in the Drosophila Hsp70 heat-shock gene [J]. Cell,1982,30(2):517-528
183. Perez-Casanova J C, Rise M L, Dixon B, et al. The immune and stress responses of Atlantic cod to long-term increases in water temperature [J]. Fish Shellfish Immunol,2008,24(5):600-609
184. Peter M E, Heufelder A E, Hengartner M O. Advances in apoptosis research [J]. Proc Natl Acad Sci USA,1997,94(24):12736-12737
185. Peters G, Faisal M, Lang T, et al. Stress caused by social interaction and its effect on susceptibility to Aeromonas hydrophila infection in rainbow trout Salmo gairdneri [J]. Dis Aquat Org,1988,4: 83-89
186. Peters G, Nuβgen A, Raabe A, et al. Social stress induces structural and functional alterations of phagocytes in rainbow trout(Oncorhynchus mykiss) [J]. Fish Shellfish lmmunol,1991,1(1):17-31
187. Piano A, Asirelli C, Caselli F, et al. Hsp70 expression in thermally stressed Ostrea edulis, a commercially important oyster in Europe [J]. Cell Stress Chaperones,2002,7(3):250-257
188. Pickering A D. Environmental stress and the survival of brown trout, Salmo trutta [J]. Freshwater Biol,1989,21(1):47-55
189. Pickering A D. Introduction:The concept of biological stress [C]//Pickering A D. Stress and Fish. London:Academic Press,1981:1-9
190. Pottinger T G. Changes in blood cortisol, glucose and lactate in carp retained in anglers'keepnets [J]. J Fish Biol,1998,53(4):728-742
191. Price B D, Calderwood S K. Ca2+ is essential for multistep activation of the heat shock factor in permeabilized cells [J]. Mol Cell Biol,1991,11(6):3365-3368
192. Procarione L S, Barry T P, Malison J A. Effects of high rearing densities and loading rates on the growth and stress responses of juvenile rainbow trout [J]. N Am J Aquacult,1999,61(2):91-96
193. Ravagnan L, Gurbuxani S, Susin S A, et al. Heat-shock protein 70 antagonizes apoptosis-inducing factor [J]. Nat Cell Biol,2001,3(9):839-843
194. Reid S G, Bernier N J, Perry S F. The adrenergic stress response in fish:control of catecholamine storage and release [J]. Comp Biochem Physiol C,1998,120(1):1-27
195. Reed K H, Larsen H J S, Linder R D, et al. Genetic variation in lysozyme activity in rainbow trout (Oncorhynchus mykiss) [J]. Aquaculture,1993,109(3-4):237-244
196. Rojtinakem J, Hirono I, Itami T. Gene expression in haemocytes of Kuruma prawn, Penaeus japonicus, in response infection with WSSV by KST approach [J]. Fish Shellfish Immunol,2002, 13(1):69-83
197. Rose T M, Schultz E R, Henikoff J G, et al. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences [J]. Nucleic Acids Res,1998,26(7):1628-1635
198. Rotllant J, Pavlidis M, Kentouri M, et al. Non-specific immune responses in the red porgy Pagrus pagrus after crowding stress [J]. Aquaculture,1997,156(3-4):279-290
199. Rotllant J, Tort L. Cortisol and glucose responses after acute stress by net handling in the sparid red porgy previously subjected to crowding stress [J]. J Fish Biol,1997,51(1):21-28
200. Ruane N M, Komen H. Measuring cortisol in the water as an indicator of stress caused by increased loading density in common carp(Cyprinus carpio) [J]. Aquaculture,2003,218(1-4):685-693
201. Ruane N M, Nolan D T, Rotllant J, et al. Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Kr(?)yer) influences the physiological response to an acute stressor [J]. Fish Shellfish Immunol,2000,10(5): 451-463
202. Sadler J, Wells R M G, Pankhurst P M, et al. Blood oxygen transport, rheology and haematological responses to confinement stress in diploid and triploid Atlantic salmon, Salmo salar [J]. Aquaculture,2000,184(3-4):349-361
203. Sahin K, Sahin N, Kucuk O. Effects of chromium, and ascorbic acid supplementation on growth, carcass traits, serum metabolites, and antioxidant status of broiler chickens reared at a high ambient temperature (32℃) [J]. Nutr Res,2003,23(2):225-238
204. Sahu S, Das B K, Pradhan J, et al. Effect of Magifera indica kernel as a feed additive on immunity and resistance to Aeromonas hydrophila in Labeo rohita fingerlings [J]. Fish Shellfish Immunol, 2007,23(1):109-118
205. Sakai D K. The assessment of the health condition of salmonids by non-specific haemolytic (SH50) activity of serum [J]. Bull Jpn soc sci fish,1983,49(10):1487-1491
206. Sakai M. Current research status of fish immunostimulants [J]. Aquaculture,1999,172(1-2):63-92
207. Sakakura Y, Koshio S, Iida Y, et al. Dietary vitamin C improves the quality of yellowtail (Seriola quinqueradiata) seedlings [J]. Aquaculture,1998,161(1-4):427-436
208. Saleh A, Srinivasula S M, Balkir L, et al. Negative regulation of the Apaf-1 apoptosome by Hsp70 [J]. Nat Cell Biol,2000,2:476-483
209. Santoro M G. Heat shock factors and the control of the stress response [J] Biochem Pharmacol, 2000,59(1) 55-63
210. Scarim A L, Heitmeier M R, Corbett J A. Heat shock inhibits cytokine-induced nitric oxide synthase expression by rat and human islets [J]. Endocrinology,1998,139(12):5050-5057
211. Schreck C B, Contreras-Sanchez W, Fitzpatrick M S. Effects of stress on fish reproduction, gamete quality, and progeny [J]. Aquaculture,2001,197(1-4):3-24
212. Selvakumar S, Geraldine P. Heat shock protein induction in the freshwater prawn Macrobrachium malcolmsonii:acclimation-influenced variations in the induction temperatures for Hsp70 [J]. Comp Biochem Physiol A,2005,140(2):209-215
213. Selye H. Stress and the general adaptation syndrome [J]. Br Med J,1950,1:1383-1392
214. Shankaran M, Yamamoto B K, Gudelsky G A. Ascorbic acid prevents 3,4-methylenedioxy methamphetamine (MDMA)-induced hydroxyl radical formation and the behavioral and neurochemical consequences of the depletion of brain 5-HT [J]. Synapse,2001,40(1):55-64
215. Shiau S Y, Hsu T S. Vitamin C requirement of grass shrimp, Penaeus monodon, as determined with L-ascorbyl-2-monophosphate [J]. Aquaculture,1994,122(4):347-357
216. Shimeno S, Shikata T, Hosokawa H, et al. Metabolic response to feeding rates in common carp, Cyprinus carpio [J]. Aquaculture,1997,151(1-4):371-377
217. Sies H. Oxidative stress:oxidants and antioxidants [J]. Exp Physiol,1997,82(2):291-295
218. Sinha A K. Colorimetric assay of catalase [J]. Anal Biochem,1972,47(2):389-394
219. Sistonen L, Sarge K D, Phillips B, et al. Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells [J]. Mol Cell Biol,1992,12(9):4104-4111
220. Sobhana K S, Mohan C V, Shankar K M. Effect of dietary vitamin C on the disease susceptibility and inflammatory response of mrigal, Cirrhinus mrigala (Hamilton) to experimental infection of Aeromonas hydrophila [J]. Aquaculture,2002,207(3-4):225-238
221. Soliman A K, Jauncey K, Roberts R J. The effect of dietary ascorbic aicd supplementation on hatchability, survival rate and fry performance in Oreochromis mossambicus (Peters) [J]. Aquaculture,1986,59(3-4):197-208
222. Sommer C V, Binkowski F P, Schalk M A, et al. Stress factors that can affect studies of drug metabolism in fish [J]. Vet Hum Toxicol,1986,28(Suppl 1):45-54
223. Song L S, Wu L T, Ni D J, et al. The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacterial challenge and naphthalin stress [J]. Fish Shellfish Immunol,2006,21(4):335-345
224. S(?)rensen J G, Kristensen T N, Loeschcke V. The evolutionary and ecological role of heat shock proteins [J]. Ecol Lett,2003,6(11):1025-1037
225. Sorger P K, Lewis M J, Pelham H R. Heat shock factor is regulated differently in yeast and HeLa cells [J]. Nature,1987,329(6134):81-84
226. Sorger P K, Pelham H R. Purification and characterization of a heat-shock element binding protein from yeast [J]. EMBO J,1987,6(10):3035-3041
227. Sovenyi J, Szakolczai J. Studies on the toxic and immunosuppressive effects of cadmium on the common carp [J]. Acta Vet Hung,1993,41(3-4):415-426
228. Sreedhar A S, Csermely P. Heat shock proteins in the regulation of apoptosis:new strategies in tumor therapy A comprehensive review [J]. Pharmacol Ther,2004,101(3):227-257
229. Srinivas G, Anto R J, Srinivas P, et al. Emodin induces apoptosis of human cervical cancer cells through poly(ADP-ribose) polymerase cleavage and activation of caspase-9 [J]. Eur J Pharmacol, 2003,473(2-3):117-125
230. Steffens W. Principles of fish nutrition [M]. New York:Ellis Horwood Press,1989:256-258
231. Stephens S M, Alkindi A Y A, Waring C P et al. Corticosteroid and thyroid responses of larval and juvenile turbot exposed to the water-soluble fraction of crude oil [J]. J Fish Biol,1997,50(5): 953-964
232. Stuart R A, Cyr D M, Neupert W. Hsp70 in mitochondrial biogenesis:From chaperoning nascent polypeptide chains to facilitation of protein degradation [J]. Experientia,1994,50(11-12): 1002-1011
233. Sumpter J P, Dye H M, Benfey T J. The effects of stress on plasma ACTH, a-MSH, and cortisol levels in salmonid fishes [J]. Gen Comp Endocrinol,1986,62(3):377-385
234. Sun L T, Chen G R, Chang C F. Acute response of blood parameters and comatose effects in salt-acclimated tilapias exposed to low temperatures [J]. J Therm Biol,1995,20(3):299-306
235. Sun P M, Liu Y T, Zhao Y G, et al. Relationship between heart damages and HSPs mRNA in persistent heat stressed broilers [J]. Agric Sci China,2007,6(2):227-233
236. Sunyer J O, Gomez E, Tort L, et al. Physiological responses and depression of humoral components of the immune system in gilthead sea bream (Sparus aurata) following daily acute stress [J]. Can J Fish Aquat Sci,1995,52(11):2339-2346
237. Tamura Y, Peng P, Liu K, et al. Immunotheraphy of tumors with autologous tumor-derived heat shock protein preparations [J]. Science,1997,278(5335):117-120
238. Tedengren M, Olsson B, Reimer O, et al. Heat pretreatment increases cadmium resistance and HSP70 levels in Baltic Sea mussels [J]. Aquatic Toxicol,2000,48(1):1-12
239. Thompson I, White A, Fletcher T C, et al. The effect of stress on the immune response of Atlantic salmon (Salmo salar L.) fed diets containing different amounts of vitamin C [J]. Aquaculture,1993, 114(1-2):1-18
240. Van Raaij M T, Pit D S, Balm P H, et al. Behavioral strategy and the physiological stress response in rainbow trout exposed to severe hypoxia [J]. Horm Behav,1996,30(1):85-92
241. Van Weerd J H, Komen J. The effects of chronic stress on growth in fish:a critical appraisal [J]. Comp Biochem Physiol A,1998,120(1):107-112
242. Vasudeva Rao Y, Das B K, Jyotyrmayee P, et al. Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila [J]. Fish Shellfish Immunol,2006, 20(3):263-273
243. Verlhac V, Gabaudan J, Obach A, et al. Influence of dietary glucan and vitamin C on non-specific and specific immune responses of rainbow trout(Oncorhynchus mykiss) [J]. Aquaculture,1996, 143(2):123-133
244. Verlhac V, Gabaudan J. Influence of vitamin C on the immune system of salmonids [J]. Aquacult Res,1994,25(1):21-36
245. Vijayan M M, Leatherland J F. High stocking density affects cortisol secretion and tissue distribution in brook charr, Salvelinus fontinalis [J]. J Endocrinol,1990,124(2):311-318
246. Waagbe R, Glette J, Raa-Nilsen E, et al. Dietary vitamin C immunity and disease resistance in Atlantic salmon (Salmo salar) [J]. Fish Physiol Biochem,1993,12(1):61-73
247. Wahli T, Verlhac V, Girling P, et al. Influence of dietary vitamin C on the wound healing process in rainbow trout(Oncorhynchus mykiss) [J]. Aquaculture,2003,225(1-4):371-386
248. Wang H H, Chung J G. Emodin-induced inhibition of growth and DNA damage in the Helicobacter pylori [J]. Curr Microbiol,1997,35(5):262-266
249. Wang X J, Kim K W, Bai S C. Effects of different dietary levels of L-ascorbyl-2-polyphosphate on growth and tissue vitamin C concentrations in juvenile olive flounder, Paralichthys olivaceus (Temminck et Schlegel) [J]. Aquae Res,2002,33(4):261-267
250. Wang Y, Xiong L, Yang K J, et al. Effect of beta-cypermethrin on GPT and GOT activities of crucian serum [J]. Agric Sci Technol,2005,6(1):20-23
251. Welch W J. How cells respond to stress [J]. Sci Am,1993,268(5):56-64
252. Welch W J. Mammalian stress response:cell physiology, structure/function of stress proteins, and implications for medicine and disease [J]. Physiol Rev,1992,72(4):1063-1081
253. Westwood J T, Clos J, Wu C. Stress-induced oligomerization and chromosomal relocalization of heat-shock factor [J]. Nature,1991,353(6347):822-827
254. Wilson R P. Absence of ascorbic acid synthesis in channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus) [J]. Comp Biochem Physiol B,1973,46(3):635-638
255. Wu C, Zimarino C, Tsai B. Transcriptional regulation of heat shock genes [C]//Morimoto R I, Tissieres A, Georgopoulos C. Stress Proteins in Biology and Medicine. New York:Cold Spring Harbor Laboratory,1990:429-442
256. Wu Y, Wan T, Zhou X, et al. Hsp70-like protein 1 fusion protein enhances induction of carcinoembryonic antigen-specific CD8+CTL response by dendritic cell vaccine [J]. Cancer Res, 2005,65(11):4947-4954
257. Xiao C, Chen S, Yuan M, et al. Expression of the 60 kDa and 71 kDa heat shock proteins and presence of antibodies against the 71 kDa heat shock protein in pediatric patients with immune thrombocytopenic purpura [J]. BMC Blood Disord,2004,4(1):1-10
258. Xie J, Liu B, Zhou Q L, et al. Effects of anthraquinone extract from rhubarb Rheum officinale Baill on the crowding stress response and growth of common carp Cyprinus carpio var. Jian [J]. Aquaculture,2008,281(1-4):5-11
259. Yamashita M, Hirayoshi K, Nagata K. Characterization of multiple members of the HSP70 family in platyfish culture cells:molecular evolution of stress protein HSP70 in vertebrates [J]. Gene,2004, 336(2):207-218
260. Yin G J, Ardo L, Thompson K D, et al. Chinese herbs (Astragalus radix and Ganoderma lucidum) enhance immune response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila [J]. Fish Shellfish Immunol,2009,26(1):140-145
261. Yin G J, Jeney G, Racz T, et al. Effect of two chinese herbs(Astragalus radix and Scutellaria radix) on non-specific immune response of tilapia, Oreochromis niloticus [J]. Aquaculture,2006,253(1-4): 39-47
262. Yin Z, Lam T J, Sin Y M. The efects of crowding stress on the non-specific immune response in fancy carp(Cyprinus carpio L.) [J]. Fish Shellfish Immunol,1995,5(7):519-529
263. Yost H J, Lindquist S. RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis [J]. Cell,1986,45(2):185-193
264. Zamzami N, Kroemer G.The mitochondrion in apoptosis:How Pandora's box opens [J]. Nat Rev Mol Cell Biol,2001,2(1):67-71
265. Zarate J, Bradley T M. Heat shock proteins are not sensitive indicators of hatchery stress in salmon [J]. Aquaculture,2003,223(1-4):175-187
266. Zhu X T, Zhao X, Burkholder W F, et al. Structural analysis of substrate binding by the molecular chaperone DnaK [J]. Science,1996,272(5268):1606-1614
267.艾春香,陈立桥,周忠良,等.维生素C、E对中华绒螯蟹生殖性能的影响[J].水产学报,2003,27(1)：62-68
268.陈琦,华修国,顾晓峰,等.复方中草药对大鼠热应激蛋白70mRNA的表达影响[J].上海交通大学学报(农业科学版),2006,24(1)：61-64
269.陈孝煊,吴志新,殷居易,等.大黄、穿心莲、板蓝根和金银花对异育银鲫免疫机能的影响[J].中国水产科学,2003,10(1)：36-40
270.陈寅儿,金珊,王国良.鲈鱼溶藻弧菌病的血液生理生化指标研究[J].台湾海峡,2005,24(1)：104-108
271.辜玲芳,侯永清,丁斌鹰,等.几种植物提取物对异育银鲫生长性能和血液生化指标的影响[J].淡水渔业,2008,38(2)：23-26
272.郭松林,关瑞章,冯建军,等.嗜水气单胞菌感染对美洲鳗鲡血.液和生化指标的影响[J].集美大学学报(自然科学版),2010,15(2)：93-97
273.侯炜,杨占秋,陈科力,等.大黄有效成分抗单纯疱疹病毒作用的实验研究[J].药物分析杂志2003,23(4)：259-262
274.胡斌,李小勤,冷向军,等.饲料Vc对草鱼生长、肌肉品质及非特异性免疫的影响[J].中国水产科学,2008,15(5)：794-800
275.胡吕江,马烈,何学梅,等.九制大黄葸醌衍生物对动物高血脂及血液流变学的影响[J].中成药,2001,23(1)：31-33
276.黄泰康.常用中药成分与药理手册[M].北京：中国医药科技出版社,1994：228
277.吉红,曹福余,于海淼.黄岑素对鲤鱼健康及生长的影响[J].安徽农业大学学报,2008,35(4)：535-539
278.姜晓峰,甄永苏.大黄素逆转肿瘤细胞多药抗药性的作用[J].药学学报,1999,34(3)：164-167
279.金抗,于东云,由田.大黄对急性肺损伤大鼠热休克蛋白70表达的影响[J].中国急救医学,2007,27(9)：827-829
280.雷爱莹,曾地刚.复方中草药对凡纳滨对虾热应激蛋白70基因表达的影响[J].广西农业科学,2008,39(6)：830-833
281.李爱华.拥挤胁迫对草鱼皮质醇、血糖及肝脏中抗坏血酸含量的影响[J].水生生物学报,1997,21(4)：384-386
282.李爱杰.水产动物营养与饲料学[M].北京：中国农业出版社,1996：55
283.李桂峰,钱沛锋,孙际佳,等.维生素C对胡子鲶Claris fuscus细胞活性和血清因子的影响[J].中山大学学报(自然科学版),2005,44(5)：75-83
284.李桂峰,钱沛锋,孙际佳,等.维生素C对胡子鲶血清免疫相关酶活性的影响[J].大连水产学院学报,2004,19(4)：301-305
285.廖昌容,陈毕生.抗生素在水产动物体内的药代动力学研究进展[J].中国兽药杂志,2004,38(3)：30-34
286.刘红柏,黄江,马爱枝,等.中草药复方添加剂对施氏鲟生长及血液生化指标的影响[J].水产学杂志,2009,22(3)：14-18
287.刘红柏,张颖,卢彤岩,等.饲料中添加中草药对鲤鱼免疫功能的影响[J].集美大学学报(自然科学版),2004,9(4)：317-321
288.陆承平.致病性嗜水气单胞菌及其所致鱼病综述[J].水产学报,1992,16(3)：282-288
289.马得莹,单安山.几种中草药抗蛋鸡热应激作用分子机制的初步研究[J].动物营养学报,2007,19(3)：283-288
290.麦康森,艾庆辉,徐玮,等.水产养殖中的环境胁迫及其预防--营养学途径[J].中国海洋大学学报,2004,34(5)：767-774
291.明建华,谢骏,刘波,等.团头鲂HSP70 cDNA的克隆、序列分析以及热应激对其mRNA表达的影响[J].中国水产科学,2009,16(5)：635-648
292.聂品.鱼类非特异性免疫研究的新进展[J].水产学报,1997,21(1)：69-73
293.饶玉才,胡宝庆,文春根.嗜水气单胞菌感染对背角无齿蚌5种免疫相关酶活力的影响[J].水生生物学报,2009,33(3)：406-412
294.史洪博,段秀梅,李树蕾,等.结核分支杆菌HSP70原核表达载体的构建和表达及蛋白纯化[J].吉林大学学报(医学版),2006,32(6)：989-992,999
295.舒筱灿,周利玲,吴和平,等.大黄素对动脉粥样硬化的干预[J].中国组织工程研究与临床康复,2007,11(34)：6856-6859
296.苏洁,蔡宏,朱玉贤.结核分枝杆菌热休克蛋白Hsp70在原核表达载体中的克隆表达与鉴定[J].中国人兽共患病学报,2006,22(8)：712-715
297.孙峰,张煜,李立德,等.感染嗜水气单胞菌对鲫鱼非特异性免疫功能的影响[J].中国海洋大学学报,2005,35(5)：815-818
298.王洪琦,邹素芬,李建国.清热复方对热休克大鼠肝、肺组织热休克蛋白基因表达的调控作用[J].广州中医药大学学报,2004,21(1)：37-39
299.王镜岩,朱圣庚,徐长法.生物化学[M].3版.北京：高等教育出版社,2006：582-583
300.王群,丁银娣,赵云龙,等.维生素C对中华绒螯蟹雄性生殖的影响[J].动物学杂志,2004,39(2)：1-5
301.王文博,汪建国,李爱华,等.拥挤胁迫后鲫鱼血液皮质醇和溶菌酶水平的变化及对病原的敏感性[J].中国水产科学,2004,11(5)：408-412
302.王文俊,吴咸中,姚智,等.大黄素、丹参素对单核细胞分泌炎性细胞因子的调节[J].中国免疫学杂志,1995,11(6)：370-372
303.王小玲.应激对黄颡鱼非特异性免疫细胞的影响[D].武汉：华中农业大学,2006：34
304.席峰.鱼类应激及其适应性生热作用[J].饲料研究,2001,10：10-12
305.谢数涛,吴任,许忠能,等.亚致死温度热休克对凡纳滨对虾的保护作用[J].中山大学学报(自然科学版),2005,44(6)：87-89
306.谢一荣,吴锐全,谢骏,等.维生素C水平对大口黑鲈抵抗嗜水气单胞菌感染的影响[J].水利渔业,2007,27(5)：102-104
307.徐小芳,罗燕,赵民,等.中药复方多糖对鸡抗氧化功能的影响[J].中国农业科学,2009,42(2)：706-713
308.徐增光,杨进波,杨杪,等.抗氧化维生素对苯并(a)芘抑制内皮细胞HSP70表达的干预作用[J].环境与职业医学,2004,21(2)：111-113
309.姚泰.生理学[M].北京：人民卫生出版社,2002：367407
310.展玉涛,刘宾,李定国,等.大黄素抗肝纤维化的作用机制[J].中华肝脏病杂志,2004,12(4)：245-246
311.展玉涛,魏红山.大黄素对大鼠四氯化碳性肝损伤保护作用的实验研究[J].中国中医药科技,2000,7(1)：30-31
312.张骏,翁福海,李会强,等.大黄素对内毒素刺激下的大鼠腹腔巨噬细胞分泌TNFα及NO的影响[J].天津医科大学学报,2001,7(2)：189-191
313.张廷军,扬振才,孙儒泳.鱼类对高密度环境的适应[J].水产科技情报,1998,25(3)：110-113
314.周利玲,舒筱灿,吴和平,等.大黄素干预鹌鹑脂肪肝病变的效果[J].中国临床康复,2006,10(32)：57-59,F0003
315.周显青,李胜利,王晓辉,等.维生素C多聚磷酸酯对小鼠肝脏脂质过氧化物和抗氧化物酶的影响[J].动物学报,2004,50(3)：370-374
316.周显青,牛翠娟,孙儒泳.维生素C和酸应激对中华鳖幼鳖血清补体C3和C4含量的影响[J].动物学报,2003,49(6)：769-774
317.周显青,乔欣.维生素C多聚磷酸酯对小鼠抗热应激能力的影响[J].动物学研究,2004,25(5)：447451
318.周玉,郭文场,杨振国,等.欧洲鳗鲡“狂游病”血液生化指标研究[J].水生生物学报,2002,26(3)：314-316
319.周玉,郭文场,杨振国,等.鱼类血液指标的研究进展[J].上海水产大学学报,2001,10(2)：163-165