乳化异氟醚对乳鼠心肌细胞缺氧/复氧损伤的保护作用及凋亡相关基因表达的影响
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摘要
目的:观察乳化异氟醚(Emulsified isofurane,EI,8 %体积分数)对培养乳鼠心肌细胞模拟缺血再灌注损伤后心肌细胞的保护作用,并对其可能的机制进行探讨,为EI以后在临床的研究及应用提供实验依据。
方法:利用原代培养的Wister乳鼠心肌细胞建立模拟心肌缺血再灌注模型,分对照组(不加药物处理,常氧条件下培养6h)、模型(H/R)组(不加药物处理,缺氧3h后复氧3h)、脂肪乳组(在培养基中加入脂肪乳干预2h,余同H/R组)和EI组(在培养基中加入EI干预2h,余同H/R组)。各组心肌细胞做相应分组处理后,利用倒置显微镜观察心肌细胞的生长状态和搏动频率,并用透射电镜观察细胞超微结构改变;XTT比色法测定细胞存活率;收集心肌细胞行超声粉碎,离心取上清,按照试剂盒说明,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性,硫代巴比妥酸显色法测定丙二醛(MDA)含量,并测定缺氧复氧后细胞培养液中乳酸脱氢酶(LDH)活性;流式细胞仪检测心肌细胞凋亡率;RT-PCR检测心肌细胞凋亡基因bcl-2 mRNA、bax mRNA的表达;Western blot定量检测Bcl-2、Bax蛋白的表达。结果:
1心肌细胞形态学观察:心肌细胞培养24h出现自发性搏动,72h后聚集成簇,呈现频率为80-100次/min的同步节律性搏动。细胞形态呈梭形或不规则三角形,伪足明显,可发生相互融合,且其超微结构完整、清楚。H/R后心肌细胞皱缩变圆,伪足减少,细胞搏幅减弱、频率减慢甚至出现停搏,超微结构出现线粒体肿胀,内质网空泡等改变。EI和脂肪乳组对H/R心肌细胞的形态和超微结构都有明显改善。
2各组心肌细胞存活率比较:与对照组比较,其余各组心肌细胞存活率均降低,差异有统计学意义(P<0.05);而与H/R组比较,EI组和脂肪乳组心肌细胞存活率明显升高,差异有统计学意义(P<0.05)。
3各组心肌细胞SOD、LDH活性与MDA含量的比较:与对照组比较,H/R组细胞SOD下降,MDA和LDH升高(均P<0.05)。EI组与H/R组比较,心肌细胞SOD活性提高,MDA和LDH含量降低(均P<0.05)。
4 EI和脂肪乳均可显著降低H/R损伤心肌细胞的凋亡率(均P<0.05),但EI组与脂肪乳组比较降低更明显(均P<0.05);EI可显著上调Bcl-2表达(P<0.05)、下调Bax表达(P<0.05);脂肪乳对Bcl-2和Bax表达无影响(P>0.05)。
结论:
1 H/R对体外培养乳鼠心肌细胞可以造成明显的损伤,表现为细胞存活率降低,心肌酶的漏出,氧自由基的产生增加,心肌细胞超微结构改变,心肌细胞凋亡率增加等。
2乳化异氟醚对体外培养乳鼠心肌细胞H/R损伤具有保护作用,表现为能提高细胞存活率,减少心肌酶的漏出,抑制氧自由基产生,维持心肌细胞超微结构完整,抑制心肌细胞凋亡,促进复氧时心肌细胞功能的恢复等。
3乳化异氟醚对体外培养乳鼠心肌细胞H/R损伤的保护作用机制可能与其抗自由基损伤、bcl-2基因表达上调和bax基因表达下调有关。
Objective: To investigate the protective effects and possible mechanism of emulsified isoflurane on myocardial cells after hypoxia-/ reoxygenation(H/R) in cultured myocardial cells from neonatal rat in order to provide experiment foundation for its later clinical reseach or application.
Methods: Myocardial hypoxia/reoxygenation model was established by culturing primary myocardial cell from neonatal rat in vitro. The myocardial cells were divided into four groups, the control group(cultured in normoxia 6h), H/R group(cultured in hypoxia 3h and reoxgenation 3h) , fat milk group(fat milk pretreatment for 2h and cultured in hypoxia 3h and reoxgenation 3h), EI group(EI pretreatment for 2h and cultured in hypoxia 3h and reoxgenation 3h). Every group cellular morphologic changes and pulsatile frequence were observed under inverted microscope and the ultrastructure of myocardial cells was observed under transmission electron microscope(TEM);Myocardial cell’s survival rate were determined by XTT;Cardiomyocytes were collected and ultrasonic-ccation for the later analysis of SOD and the content of MDA,the activities of plasma superoxide dismutase (SOD) were measured by method of xanthine oxidase and the contents of serum Malonicaldehyde (MDA) by the method of thiobarbituric acid;The activities of lactate dehydrogenase (LDH) in cell-cultured solution after H-R injury were measured according to relevant kits; The apoptotic rate of myocardial cells was determined by flow cytometry; The expression of bcl-2 mRNA and bax mRNA were detected by using RT-PCR method; The expression of Bcl-2/Bax protein were observed by Western blot.
Results:
1 The morphologic changes of cardiomyocytes: After H/R procedure cardiomyocytes beated unregularily and powerlessly,sells contracted and tended to get round in shape ,cellular pseudopods decreased in H/R,EI ,fat milk groups compared with control group. The ultrastrctural changes of cardiomyocytes were improved in EI,fat milk groups compared with H/R.
2 The survival rate of myocardial cell:Compared with control group,the rate decreased in H/R,EI,fat milk groups(p<0.05).but compared with H/R group,the rate increased in EI and fat milk groups.
3 SOD activities and LDH,MDA concentration: Compared with control group,SOD activity decreased and MDA,LDH increased in H/R group (p<0.05). Compared with H/R group, SOD activity increased, MDA and LDH decreased (p<0.05)in EI and fat milk groups.
4 The apoptotic rate and the expression of correlated apoptotic gene Both EI and fat milk groups could significantly decrease the apoptotic rate (P< 0.05),while EI shoud have stronger effect(P<0.05). EI group signify -cantly up-regulated Bcl-2 expression and down-regulated Bax expression (P<0.05) while fat milk group can’t(P< 0.05).
Conclusion
1 H/R injured cultured myocardial cells from neonatal rat markedly,including the decreasing of myocardial cell’s survival rate, the increasing of serum myocardial enzyme leakage and myocardial MDA,the elevation of apoptotic rate ,injury of myocardial ultrastructure.
2 EI pretreatment had the protective effects on cultured myocardial cells from neonatal rat injured by H-R.,which included reduction of myocardial cell’s survival rate,the reduction of the leakage of myocardial enzyme,reduction of oxygen-derived free radials,protection of myocardial ultrastructure,inhibition of apoptosis, better recovery of cardiac function during reoxygenation.
3 EI pretreatment had the protective effects on cultured myocardial cells from neonatal rat injured by H-R. The possible mechanism may be that: one is by reducing Oxygen Free Radical,the other is by inhibiting myocardial apoptosis through up-regulating Bcl-2 expression and down- regulating Bax expression .
方法:利用原代培养的Wister乳鼠心肌细胞建立模拟心肌缺血再灌注模型,分对照组(不加药物处理,常氧条件下培养6h)、模型(H/R)组(不加药物处理,缺氧3h后复氧3h)、脂肪乳组(在培养基中加入脂肪乳干预2h,余同H/R组)和EI组(在培养基中加入EI干预2h,余同H/R组)。各组心肌细胞做相应分组处理后,利用倒置显微镜观察心肌细胞的生长状态和搏动频率,并用透射电镜观察细胞超微结构改变;XTT比色法测定细胞存活率;收集心肌细胞行超声粉碎,离心取上清,按照试剂盒说明,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性,硫代巴比妥酸显色法测定丙二醛(MDA)含量,并测定缺氧复氧后细胞培养液中乳酸脱氢酶(LDH)活性;流式细胞仪检测心肌细胞凋亡率;RT-PCR检测心肌细胞凋亡基因bcl-2 mRNA、bax mRNA的表达;Western blot定量检测Bcl-2、Bax蛋白的表达。结果:
1心肌细胞形态学观察:心肌细胞培养24h出现自发性搏动,72h后聚集成簇,呈现频率为80-100次/min的同步节律性搏动。细胞形态呈梭形或不规则三角形,伪足明显,可发生相互融合,且其超微结构完整、清楚。H/R后心肌细胞皱缩变圆,伪足减少,细胞搏幅减弱、频率减慢甚至出现停搏,超微结构出现线粒体肿胀,内质网空泡等改变。EI和脂肪乳组对H/R心肌细胞的形态和超微结构都有明显改善。
2各组心肌细胞存活率比较:与对照组比较,其余各组心肌细胞存活率均降低,差异有统计学意义(P<0.05);而与H/R组比较,EI组和脂肪乳组心肌细胞存活率明显升高,差异有统计学意义(P<0.05)。
3各组心肌细胞SOD、LDH活性与MDA含量的比较:与对照组比较,H/R组细胞SOD下降,MDA和LDH升高(均P<0.05)。EI组与H/R组比较,心肌细胞SOD活性提高,MDA和LDH含量降低(均P<0.05)。
4 EI和脂肪乳均可显著降低H/R损伤心肌细胞的凋亡率(均P<0.05),但EI组与脂肪乳组比较降低更明显(均P<0.05);EI可显著上调Bcl-2表达(P<0.05)、下调Bax表达(P<0.05);脂肪乳对Bcl-2和Bax表达无影响(P>0.05)。
结论:
1 H/R对体外培养乳鼠心肌细胞可以造成明显的损伤,表现为细胞存活率降低,心肌酶的漏出,氧自由基的产生增加,心肌细胞超微结构改变,心肌细胞凋亡率增加等。
2乳化异氟醚对体外培养乳鼠心肌细胞H/R损伤具有保护作用,表现为能提高细胞存活率,减少心肌酶的漏出,抑制氧自由基产生,维持心肌细胞超微结构完整,抑制心肌细胞凋亡,促进复氧时心肌细胞功能的恢复等。
3乳化异氟醚对体外培养乳鼠心肌细胞H/R损伤的保护作用机制可能与其抗自由基损伤、bcl-2基因表达上调和bax基因表达下调有关。
Objective: To investigate the protective effects and possible mechanism of emulsified isoflurane on myocardial cells after hypoxia-/ reoxygenation(H/R) in cultured myocardial cells from neonatal rat in order to provide experiment foundation for its later clinical reseach or application.
Methods: Myocardial hypoxia/reoxygenation model was established by culturing primary myocardial cell from neonatal rat in vitro. The myocardial cells were divided into four groups, the control group(cultured in normoxia 6h), H/R group(cultured in hypoxia 3h and reoxgenation 3h) , fat milk group(fat milk pretreatment for 2h and cultured in hypoxia 3h and reoxgenation 3h), EI group(EI pretreatment for 2h and cultured in hypoxia 3h and reoxgenation 3h). Every group cellular morphologic changes and pulsatile frequence were observed under inverted microscope and the ultrastructure of myocardial cells was observed under transmission electron microscope(TEM);Myocardial cell’s survival rate were determined by XTT;Cardiomyocytes were collected and ultrasonic-ccation for the later analysis of SOD and the content of MDA,the activities of plasma superoxide dismutase (SOD) were measured by method of xanthine oxidase and the contents of serum Malonicaldehyde (MDA) by the method of thiobarbituric acid;The activities of lactate dehydrogenase (LDH) in cell-cultured solution after H-R injury were measured according to relevant kits; The apoptotic rate of myocardial cells was determined by flow cytometry; The expression of bcl-2 mRNA and bax mRNA were detected by using RT-PCR method; The expression of Bcl-2/Bax protein were observed by Western blot.
Results:
1 The morphologic changes of cardiomyocytes: After H/R procedure cardiomyocytes beated unregularily and powerlessly,sells contracted and tended to get round in shape ,cellular pseudopods decreased in H/R,EI ,fat milk groups compared with control group. The ultrastrctural changes of cardiomyocytes were improved in EI,fat milk groups compared with H/R.
2 The survival rate of myocardial cell:Compared with control group,the rate decreased in H/R,EI,fat milk groups(p<0.05).but compared with H/R group,the rate increased in EI and fat milk groups.
3 SOD activities and LDH,MDA concentration: Compared with control group,SOD activity decreased and MDA,LDH increased in H/R group (p<0.05). Compared with H/R group, SOD activity increased, MDA and LDH decreased (p<0.05)in EI and fat milk groups.
4 The apoptotic rate and the expression of correlated apoptotic gene Both EI and fat milk groups could significantly decrease the apoptotic rate (P< 0.05),while EI shoud have stronger effect(P<0.05). EI group signify -cantly up-regulated Bcl-2 expression and down-regulated Bax expression (P<0.05) while fat milk group can’t(P< 0.05).
Conclusion
1 H/R injured cultured myocardial cells from neonatal rat markedly,including the decreasing of myocardial cell’s survival rate, the increasing of serum myocardial enzyme leakage and myocardial MDA,the elevation of apoptotic rate ,injury of myocardial ultrastructure.
2 EI pretreatment had the protective effects on cultured myocardial cells from neonatal rat injured by H-R.,which included reduction of myocardial cell’s survival rate,the reduction of the leakage of myocardial enzyme,reduction of oxygen-derived free radials,protection of myocardial ultrastructure,inhibition of apoptosis, better recovery of cardiac function during reoxygenation.
3 EI pretreatment had the protective effects on cultured myocardial cells from neonatal rat injured by H-R. The possible mechanism may be that: one is by reducing Oxygen Free Radical,the other is by inhibiting myocardial apoptosis through up-regulating Bcl-2 expression and down- regulating Bax expression .
引文
[1] 孙海峰, 王泉云, 梁小民, 等. 乳化异氟醚腹腔注射对大鼠的麻醉作用和安全性. 中华麻醉学杂志[J], 2004, 24(1):51-53.
[2] 马汉祥, 李杰, 杨宗斌, 等. 犬静脉注射乳化异氟醚与异丙酚麻醉的效果。中华麻醉学杂志[J], 2006, 26(9):862-863.
[3] 杨经文, 陈燕, 高安量, 等. 乳化异氟醚与芬太尼麻醉作用的相互影响. 华西药学杂志[J], 2007,22(5):527-529.
[4] 周建新, 罗南富, 刘进, 等. 序贯法测定乳化挥发性麻醉药在大鼠静脉诱导中的半数有效量. 麻醉与监护论坛, 2002, 9:24-26.
[5] 陈永权, 金孝炬, 戴泽平, 等. 乳化异氟醚预处理对兔缺血再灌注心肌血流动力学的影响[J]. 国际麻醉学与复苏杂志, 2006, 27(4):224-226.
[6] 刘陕岭, 刘进. 乳化异氟醚和脂肪乳对大鼠心脏缺血再灌注损伤的影响. 华西药学杂志, 2007, 22(5): 525-527.
[7] Na HS, Kim YI, Yoon YW, et al. Ventricular premature beat-driven intermittent restoration of coronary blood flow reduces the incidence of reperfusion-induced ventricular fibrillation in a cat model of regional ischemia [J]. Am Heart J, 1996, 132:78-83.
[8] Tsang A, Hausenloy DJ, Mocanu MM, et al. Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway [J]. Circ Res, 2004,95(3):230-232.
[9] 王娜, 关鹏, 段相林, 等. 新生大鼠心肌细胞原代培养方法的改进[J]. 河北师范大学学报,2007,31(2):256-259.
[10] 赵晓辉, 彭毅志, 王元元, 等. 缺氧复氧心肌细胞中 HSP90 对 AKT 表达的调控作用[J]. 现代生物医学进展, 2007,7(4):521-524.
[11] Sonpaa MH, Field LJ. Survey of studies examing mammalian cardiomyocytes DNA systhesis. Circ Res, 1998,83:15-26.
[12] 李元建, 体外培养大白鼠乳鼠心肌细胞缺氧/在给氧损伤实验法,见:徐叔云,卞如濂, 陈修主编. 药理试验方法学. 第二版. 北京:人民卫生出版社, 1991.942-944.
[13] Edelson JD, Shanoon JM, Mason RJ, et al. A maker of alveolar type II cell differentiation. AM Rev Respir Dis, 1988,138:1268(5)-1275.
[14] Van de Velde M, DeWolff M, Leather HA, et a1. Effects of lipids on thefunctional and metabolic recovery from global myocardial stunning in isolated rabbit hearts[J]. Cardiov Rese, 2000, 48(1):129- 137.
[15] Jiang MT, Nakae Y, Ljubkovic M, et al. Isoflurane activates human cardiac mitochondrial adenosine triphosphate-sensitive K+ channels reconstituted in lipid bilayers. Anesth Analg, 2007,105(4):926-932.
[16] Patel HH, Tsutsumi YM, Head BP, et al. Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1. FASEB J, 2007, 21(7):1565-1574.
[17] Lee MC, Chen CH, Kuo MC, et al. Isoflurane preconditioning-induced cardio- protection in patients undergoing coronary artery bypass grafting. Eur J Anaesthesiol, 2006,23(10):841-847.
[18] Tyther R, Fanning N,Halligan M, et al.The effect of the anaesthetic agent isoflurane on the rate of neutrophil apoptosis in vitro[J].Med Sci,2001; 170(1):41-44.
[19] Wise-Faberowski L, Raizada MK, Summners C.Oxygen and glucose deprivation- induced neuronal apoptosis is attenuated by halothane and isoflurane[J]. AnesthAnalg,2001;93(5):1281-1287.
[20] 张光明,瞿 涤,蒋 豪,等. 异氟醚对人单核细胞株 THP-1 细胞凋亡的影响[J].中国临床医学,2005; 12 (4):651-653.
[21] Wang MD C,Neff DA,Krolikowski JG,et al.The influence of B-Cell Lymphoma 2 protein,an antiapoptotic regulator of mitochondrial permeability transition,on isoflurane induced and ischemic postconditioning in rabbits[J]. AnesthAnalg, 2006; 102(5):1355-1360.
[22] Qin F, Shite J, Mao W, et al. Selegiline atenuates cardiac oxidative stress and apoptosis in heart failure: association with improvement of cardia function[J]. Pharmacol,2003;461(3):149-158.
[23] Hofstaetter B, Taimor G, Inserte J,et al. Inhibition of apoptotic responses after ischemic stress in isolated hearts and cardiomyocytes[J].Basic Res Cardiol,2002; 97(6):479-488.
[24] Hochhauser E, Kivity S, Offen D,et al. Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice[J].Physiol Heart Circ Physiol, 2003;284(6):H2351-2359.
[1] Mignotte B,Vayssiere JL.Mitochondria and apoptosis.Eur.J Biochem,I998, 252(1):1-15.
[2] KnudsonCM, Korsmeyer SJ. Bcl-2and bax function independently to regulate cell death. Nat Genet, 1997,16(4):358-363.
[3] Antonsson B, Conti F, Ciavatta A, et al. Inhibition of bax channel forming activity by Bcl-2.Science,1997,277(5324):370-372.
[4] Naumovski L, cleary ML.The p53-binding 53 BP2 also inferacts with bcl-2 and impedes cell cycle progression at G2/M. Mol Cell Biol,1996,16:3884-3892
[5] Saleh A,Srinivasula SM, Balkir L,Robbins PD, Alnemri ES. Negative regulation of the Apaf-1apoptosome by Hsp70. Nat Cell Biol,2000,2(8):476–483.
[6] Skulachev VP. Cytochrome C in the apoptotic and antioxidant cascades. FEBS Lett, 1998,423(3):275-280
[7] Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res, 1996, 79(5):949-956.
[8] 穆军升.NO 与心血管疾病细胞凋亡. 国外医学(生理,病理科学与临床分册). 2001, 21(5): 389-391.
[9] Chakrabarti S, Hoque AN, Karrnazyn M. A rapid ischemia induced apoptosis in isolated rat hearts and its attenuation by the sodium-hydrogen exchang inhibitor HoE 642(cariporide). J Mol Cell Cardiol,1997,29(11):3169-3174
[10] 吴青,陶宏凯,陶大昌,等.灌注诱导心肌细胞凋亡及凋亡相关基因表达的研究.中国心血管病研究杂志,2004;2(11) :905.
[11] 汤晓琴, 赵建洪, 张正义等. 纳洛酮对急性缺血再灌注心肌细胞 bcl-2 蛋白和肿瘤坏死因子-α 表达的影响.中国危重病急救医学,2005;17(7):430-432.
[12] 尹雪莲, 成蓓. Urocortin 调控 bcl-2 家族与大鼠缺氧/复氧心肌细胞凋亡. 微循环学杂志, 2005,15(4):22-24.
[13] Kumar D,Jugdutt Bodhl. Apoptosis andoxidants in the heart. J Lab Clin med, 2003; 142(5):288-297.
[14] Borutaite V,Brown GC. Mitochondria in apoptosis of ischemic heart. FEBS Lett, 2003;541(1-3):1-5.
[15] Yang M,Sui DJ,Zhu S. Effects of the total flavones of propolis(TFP)on Fas,bax andbcl-2 protein expression in myocardial ischemic-reperfused injury rats. Chinese PharmacologicalBulletin, 2005,21(7):799-803.
[16] Liu ZX,Hu LC,Liu XC,et al.Effects of radix ginseng rubra on cardiomyocyte apoptosis and bcl2 gene expression induced by ischemia and reperfusion in vivo in rats.J Huazhong Univ sci tech [Health Sci],2002,31 (4):376 -378
[17] Xie Z, Koyama T, Suzuki J, et al. Coronary reperfusion following ischemia: different expressionof bcl-2 and bax proteins, and cardiomyocyte apopotosis. Jpn Heart J,2001, 42 (6): 759-70.
[18] 顾国嵘, 黄培志, 童朝阳. 血塞通预处理对心肌缺血再灌注损伤的早期保护作用. 中国急救医学,2005,25(4):264-265.
[19] 杨立明,张东庆,刘正湘. Carvedilol 对大鼠实验性缺血再灌注肌细胞凋亡的影响. Chinese journalof histochemistry and cytochemistry, 2006,9(2):145-148.
[20] 蓝荣芳, 李志刚, 刘正湘. 人参皂甙 Rb 对大鼠缺血再灌注心肌细胞 Bcl-2、Bax、Bad、Fas 基因表达的影响. 中国组织化学与细胞化学杂志, 2002,11(2): 149-152,232.
[21] Fan GC, Ren XP,Qian J,et al. Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury. Circulation. 2005,111(14): 1792-1799.
[2] 马汉祥, 李杰, 杨宗斌, 等. 犬静脉注射乳化异氟醚与异丙酚麻醉的效果。中华麻醉学杂志[J], 2006, 26(9):862-863.
[3] 杨经文, 陈燕, 高安量, 等. 乳化异氟醚与芬太尼麻醉作用的相互影响. 华西药学杂志[J], 2007,22(5):527-529.
[4] 周建新, 罗南富, 刘进, 等. 序贯法测定乳化挥发性麻醉药在大鼠静脉诱导中的半数有效量. 麻醉与监护论坛, 2002, 9:24-26.
[5] 陈永权, 金孝炬, 戴泽平, 等. 乳化异氟醚预处理对兔缺血再灌注心肌血流动力学的影响[J]. 国际麻醉学与复苏杂志, 2006, 27(4):224-226.
[6] 刘陕岭, 刘进. 乳化异氟醚和脂肪乳对大鼠心脏缺血再灌注损伤的影响. 华西药学杂志, 2007, 22(5): 525-527.
[7] Na HS, Kim YI, Yoon YW, et al. Ventricular premature beat-driven intermittent restoration of coronary blood flow reduces the incidence of reperfusion-induced ventricular fibrillation in a cat model of regional ischemia [J]. Am Heart J, 1996, 132:78-83.
[8] Tsang A, Hausenloy DJ, Mocanu MM, et al. Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway [J]. Circ Res, 2004,95(3):230-232.
[9] 王娜, 关鹏, 段相林, 等. 新生大鼠心肌细胞原代培养方法的改进[J]. 河北师范大学学报,2007,31(2):256-259.
[10] 赵晓辉, 彭毅志, 王元元, 等. 缺氧复氧心肌细胞中 HSP90 对 AKT 表达的调控作用[J]. 现代生物医学进展, 2007,7(4):521-524.
[11] Sonpaa MH, Field LJ. Survey of studies examing mammalian cardiomyocytes DNA systhesis. Circ Res, 1998,83:15-26.
[12] 李元建, 体外培养大白鼠乳鼠心肌细胞缺氧/在给氧损伤实验法,见:徐叔云,卞如濂, 陈修主编. 药理试验方法学. 第二版. 北京:人民卫生出版社, 1991.942-944.
[13] Edelson JD, Shanoon JM, Mason RJ, et al. A maker of alveolar type II cell differentiation. AM Rev Respir Dis, 1988,138:1268(5)-1275.
[14] Van de Velde M, DeWolff M, Leather HA, et a1. Effects of lipids on thefunctional and metabolic recovery from global myocardial stunning in isolated rabbit hearts[J]. Cardiov Rese, 2000, 48(1):129- 137.
[15] Jiang MT, Nakae Y, Ljubkovic M, et al. Isoflurane activates human cardiac mitochondrial adenosine triphosphate-sensitive K+ channels reconstituted in lipid bilayers. Anesth Analg, 2007,105(4):926-932.
[16] Patel HH, Tsutsumi YM, Head BP, et al. Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1. FASEB J, 2007, 21(7):1565-1574.
[17] Lee MC, Chen CH, Kuo MC, et al. Isoflurane preconditioning-induced cardio- protection in patients undergoing coronary artery bypass grafting. Eur J Anaesthesiol, 2006,23(10):841-847.
[18] Tyther R, Fanning N,Halligan M, et al.The effect of the anaesthetic agent isoflurane on the rate of neutrophil apoptosis in vitro[J].Med Sci,2001; 170(1):41-44.
[19] Wise-Faberowski L, Raizada MK, Summners C.Oxygen and glucose deprivation- induced neuronal apoptosis is attenuated by halothane and isoflurane[J]. AnesthAnalg,2001;93(5):1281-1287.
[20] 张光明,瞿 涤,蒋 豪,等. 异氟醚对人单核细胞株 THP-1 细胞凋亡的影响[J].中国临床医学,2005; 12 (4):651-653.
[21] Wang MD C,Neff DA,Krolikowski JG,et al.The influence of B-Cell Lymphoma 2 protein,an antiapoptotic regulator of mitochondrial permeability transition,on isoflurane induced and ischemic postconditioning in rabbits[J]. AnesthAnalg, 2006; 102(5):1355-1360.
[22] Qin F, Shite J, Mao W, et al. Selegiline atenuates cardiac oxidative stress and apoptosis in heart failure: association with improvement of cardia function[J]. Pharmacol,2003;461(3):149-158.
[23] Hofstaetter B, Taimor G, Inserte J,et al. Inhibition of apoptotic responses after ischemic stress in isolated hearts and cardiomyocytes[J].Basic Res Cardiol,2002; 97(6):479-488.
[24] Hochhauser E, Kivity S, Offen D,et al. Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice[J].Physiol Heart Circ Physiol, 2003;284(6):H2351-2359.
[1] Mignotte B,Vayssiere JL.Mitochondria and apoptosis.Eur.J Biochem,I998, 252(1):1-15.
[2] KnudsonCM, Korsmeyer SJ. Bcl-2and bax function independently to regulate cell death. Nat Genet, 1997,16(4):358-363.
[3] Antonsson B, Conti F, Ciavatta A, et al. Inhibition of bax channel forming activity by Bcl-2.Science,1997,277(5324):370-372.
[4] Naumovski L, cleary ML.The p53-binding 53 BP2 also inferacts with bcl-2 and impedes cell cycle progression at G2/M. Mol Cell Biol,1996,16:3884-3892
[5] Saleh A,Srinivasula SM, Balkir L,Robbins PD, Alnemri ES. Negative regulation of the Apaf-1apoptosome by Hsp70. Nat Cell Biol,2000,2(8):476–483.
[6] Skulachev VP. Cytochrome C in the apoptotic and antioxidant cascades. FEBS Lett, 1998,423(3):275-280
[7] Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res, 1996, 79(5):949-956.
[8] 穆军升.NO 与心血管疾病细胞凋亡. 国外医学(生理,病理科学与临床分册). 2001, 21(5): 389-391.
[9] Chakrabarti S, Hoque AN, Karrnazyn M. A rapid ischemia induced apoptosis in isolated rat hearts and its attenuation by the sodium-hydrogen exchang inhibitor HoE 642(cariporide). J Mol Cell Cardiol,1997,29(11):3169-3174
[10] 吴青,陶宏凯,陶大昌,等.灌注诱导心肌细胞凋亡及凋亡相关基因表达的研究.中国心血管病研究杂志,2004;2(11) :905.
[11] 汤晓琴, 赵建洪, 张正义等. 纳洛酮对急性缺血再灌注心肌细胞 bcl-2 蛋白和肿瘤坏死因子-α 表达的影响.中国危重病急救医学,2005;17(7):430-432.
[12] 尹雪莲, 成蓓. Urocortin 调控 bcl-2 家族与大鼠缺氧/复氧心肌细胞凋亡. 微循环学杂志, 2005,15(4):22-24.
[13] Kumar D,Jugdutt Bodhl. Apoptosis andoxidants in the heart. J Lab Clin med, 2003; 142(5):288-297.
[14] Borutaite V,Brown GC. Mitochondria in apoptosis of ischemic heart. FEBS Lett, 2003;541(1-3):1-5.
[15] Yang M,Sui DJ,Zhu S. Effects of the total flavones of propolis(TFP)on Fas,bax andbcl-2 protein expression in myocardial ischemic-reperfused injury rats. Chinese PharmacologicalBulletin, 2005,21(7):799-803.
[16] Liu ZX,Hu LC,Liu XC,et al.Effects of radix ginseng rubra on cardiomyocyte apoptosis and bcl2 gene expression induced by ischemia and reperfusion in vivo in rats.J Huazhong Univ sci tech [Health Sci],2002,31 (4):376 -378
[17] Xie Z, Koyama T, Suzuki J, et al. Coronary reperfusion following ischemia: different expressionof bcl-2 and bax proteins, and cardiomyocyte apopotosis. Jpn Heart J,2001, 42 (6): 759-70.
[18] 顾国嵘, 黄培志, 童朝阳. 血塞通预处理对心肌缺血再灌注损伤的早期保护作用. 中国急救医学,2005,25(4):264-265.
[19] 杨立明,张东庆,刘正湘. Carvedilol 对大鼠实验性缺血再灌注肌细胞凋亡的影响. Chinese journalof histochemistry and cytochemistry, 2006,9(2):145-148.
[20] 蓝荣芳, 李志刚, 刘正湘. 人参皂甙 Rb 对大鼠缺血再灌注心肌细胞 Bcl-2、Bax、Bad、Fas 基因表达的影响. 中国组织化学与细胞化学杂志, 2002,11(2): 149-152,232.
[21] Fan GC, Ren XP,Qian J,et al. Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury. Circulation. 2005,111(14): 1792-1799.