新型炎症相关基因CNT2b的发现、克隆及功能初探
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摘要
炎症和免疫反应是一种常见的病理过程,大多数的人类疾病均与此有关。除了人们所熟知的感染性疾病外,风湿性关节炎、类风湿疾病、系统性红斑狼疮、肝纤维化、动脉粥样硬化、心肌梗塞、阿尔茨海默病以及恶性肿瘤等多种疾病也与炎症密切相关。炎症免疫因素参与了这些疾病的发生、发展及转归整个过程。巨噬细胞是机体免疫系统的一种重要细胞,不仅具有很强的吞噬功能,而且是主要的抗原提呈细胞,在多种炎症和免疫反应中起关键作用。活化的巨噬细胞可分泌多种生物活性物质,如一氧化氮、白介素-1、肿瘤坏死因子α、活性氧以及前列腺素、白三烯等花生四烯酸代谢产物。诱生型环氧酶(cyclooxygenase,COX)—COX-2可大量表达于巨噬细胞中。种种研究迹象表明,COX-2并非专一性地参加炎症反应,还在保护胃粘膜、维护肾脏功能、学习记忆等方面发挥重要作用,可能还存在其它亚型。众多的炎症因子在炎症反应的不同时期组成复杂的网络,相互作用,共同调控炎症反应的的进程和预后。巨噬细胞在炎症免疫反应中所起的重要作用,且其在诱导状态下能大量表达COXs,因此我们拟针对COX的保守序列设计简并引物,选择急性炎症诱导下的巨噬细胞cDNA作为模板克隆新型COX亚型或炎症相关性基因,并对其表达特征和功能进行初步研究。
方法
1.分析不同物种、不同亚型的COX的保守序列,设计简并引物。
2.采用腹腔注射酵母多糖A的方法建立SD大鼠急性腹膜炎模型,采集腹腔巨噬细胞,提取总RNA,进行RT-PCR,扩增产物纯化后连接T载体,转化DH5α细菌,抽提质粒,测序。
3.选取新型序列,进行3’,5’快速末端反应,获取全长基因序列。
4.采用Realtime RT-PCR法分别检测新基因CNT2 mRNA、CNT2b mRNA在大鼠不同脏器组织中的表达情况,以及在酵母多糖诱导的急性腹膜炎大鼠腹腔巨噬细胞中表达的时相特征。
5.应用Realtime RT-PCR法观察抗炎药物阿司匹林(12mg/kg)、塞来昔布(0.13mg/kg)和泼尼松(0.4mg/kg)对大鼠腹腔巨噬细胞中CNT2b mRNA和CNT2 mRNA表达的影响。
6.针对CNT2b序列设计siRNA干扰序列,采用脂质体转染体外培养NR8383大鼠巨噬细胞;Realtime RT-PCR法检测干扰后细胞中炎症相关因子IL-1α、IL-1β、IL-6、TNF-α、COX-2、IL-10以及CNT2、CNT2b等分子的mRNA表达情况。
结果
1.不同物种的COX-1、COX-2和COX-3序列中存在两段氨基酸保守序列。
2.从大鼠腹腔巨噬细胞中发现并克隆了一个新基因,全长13330nt,为一mRNA序列,对应DNA位于大鼠第三号染色体3q35区。经BLAST分析与序列比对,该基因与Na+依赖性核苷转运载体(Na+/nucleoside cotransporter, CNT)2有较高同源性,保留了CNT2的第1、7~14、16、17内含子;并且,在CNT2的最后一个(第18个)外显子后,又附加了一段3169nt长的序列。由于该基因和CNT2均包含加尾信号和polyA尾巴,均为完整的转录产物,故确定为CNT2的剪接异构体,已提交Genbank数据库并获得登录号:EU032627.2,重新命名为CNT2b。
3. CNT2 mRNA在全身各器官均有表达,其中在心脏、肝脏、平滑肌、脾脏表达较高;CNT2b mRNA在正常器官中表达量远远少于CNT2 mRNA,其中心脏、平滑肌、肝脏表达稍高。
4. CNT2b mRNA在腹膜炎大鼠腹腔巨噬细胞中的表达具有显著时相特征。注射酵母多糖后,其表达显著升高,至2h时达到最高点;随后,表达量逐渐降低,至24h时基本恢复至0.5h时的水平;致炎后36h,表达量再次明显升高,至第48h时又降至36h时的47.52%。CNT2 mRNA在腹膜炎大鼠腹腔巨噬细胞中的表达也具有类似特征,其时相变化幅度远少于CNT2b mRNA。
5.三种抗炎药物阿司匹林、泼尼松、塞来昔布均能明显降低CNT2b mRNA表达量。在临床使用剂量下,泼尼松作用最强,阿司匹林次之,塞来昔布最弱。
6.抑制CNT2b基因表达后NR8383细胞中IL-1βmRNA,IL-6 mRNA,TNF-αmRNA,COX-2 mRNA的表达均显著升高,IL-10 mRNA的表达明显降低。
结论
1.在大鼠腹腔巨噬细胞中发现并克隆了一个新基因CNT2b。
2. CNT2b属于炎症诱导性高表达基因,在正常组织中很少表达,而在急性炎症刺激的巨噬细胞中表达显著增加。在大鼠急性腹膜炎过程中,腹腔巨噬细胞CNT2b mRNA的表达具有明显时相性,呈双峰特征。抗炎药物能明显抑制CNT2b mRNA的表达。
3.抑制CNT2b的表达能促进多种促炎因子的表达,降低抗炎因子IL-10的表达。
4. CNT2b参与了炎症反应的调控过程,其功能与抑制炎症反应有关。
Inflammation and immune response is one of the most common pathological processes, and various human diseases are related to it. In addition to well-known infectious diseases, rheumatoid arthritis, rheumatoid disease, systemic lupus erythematosus, liver fibrosis, atherosclerosis, myocardial infarction, Alzheimer's disease, as well as a variety of malignant diseases are closely related to inflammation, which involved in the occurrence, development and prognosis in the whole disease process. Macrophage is an important kind of cell in immune system, which not only has a strong phagocytic function, but also be main antigen-presenting cell, and play a key role in a variety of inflammatory and immune responses. Activated macrophage can secrete a variety of bioactive substances, such as nitric oxide, interleukine-1, tumor necrosis factor-α, reactive oxygen species. Inducible cyclooxygenase (COX), COX-2 can be highly expressed in macrophage. Many studies have suggested that COX-2 is not specific to participate in inflammatory response, but play an important role in protection of gastric mucosa, maintenance of renal function, learning and memory, so other subtypes of COX might exist. A large number of inflammatory factors form the complex inflammation-immune network, which interact, co-regulate and control the process of inflammatory response and prognosis at different stages. To search specific factors for the regulation of inflammatory response will be far-reaching significance to a deep understanding of the pathophysiological mechanisms of inflammation, as well as the treatment of inflammatory diseases.
Methods
1. To design the degenerate primers base on the consensus sequence of different cyclooxytenases isoforms from various species.
2. Acute peritonitis model was established in rats using intraperitoneal injection of zymosan A. Total RNA was extracted from peritoneal macrophages, and then purified PCR products were ligated into T vector and sequenced with automated technology.
3. Full-length sequence of the novel gene was obtained by 3’and 5’rapid amplification of cDNA ends technique.
4. The mRNA expression level of CNT2 and the novel gene CNT2b in different tissues in rats and temporal expression pattern in macrophages from the zymosan A-induced acute peritonitis of rat were detected by Realtime RT-PCR technique.
5. The effects of anti-inflammatory drug aspirin (12mg/kg), celecoxib (0.13mg/kg) and prednisone (0.4mg/kg) on CNT2b mRNA and CNT2 mRNA expression in rat peritoneal macrophages were measured by Realtime RT-PCR.
6. siRNA interference sequence was designed according to CNT2b sequence and transfected into NR8383 rat macrophages using liposomes in vitro.
7. After siRNA interference, mRNA expression of inflammation related factors in NR8383 rat macrophages, such as IL-1α, IL-1β, IL-6, TNF-α, CNT2, CNT2b, COX-2, IL-10 were measured by Realtime RT-PCR.
Results
1. Two conservative amino acid sequences were observed in different species of COX-1, COX-2 and COX-3 sequences.
2. A novel mRNA sequence, 13330nt long, was found and cloned from rat peritoneal macrophages, which corresponding DNA is located at rat chromosome 3q35. BLAST analysis and sequence alignment indicated that the novel gene had high homology with Na+-dependent nucleoside transporters (Na+/nucleoside cotransporter, CNT) 2, which retained the intron 1,7-14,16,17 and a 3197nt sequences was appendixed to the last exon of CNT2. Since the novel gene and CNT2 were both complete transcriptions, which contained tailing signal and poly A tail, it was identified as the splicing isomers of CNT2 and named as CNT2b.
3. CNT2 mRNA was expressed in different tissues and the level was higher in heart, liver, skeletal muscle and spleen; CNT2b mRNA expression level in normal organs was far less than that of CNT2, and the level was slightly higher in heart, skeletal muscle and liver.
4. The significant phase characteristics of CNT2b mRNA expression was observed in peritoneal macrophages from peritonitis rat. After zymosan injection, CNT2b mRNA expression level was significantly increased and reached the peak point at 2h; subsequently, the expression level was gradually decreased, and returned to their levels at 0.5h until 24h. At 36th hour after inflammatory stimulation, CNT2b mRNA expression level was significantly increased again. The expression level of CNT2b mRNA was decreased by about 53% at 48th hour after inflammatory stimulation by comparison of that at 36th hour.
5. After treated with aspirin, prednisone and celecoxib, respectively, CNT2b mRNA expression decreased significantly, in which the effect of prednisone was strongest and celecoxib weakest.
6. Suppression of CNT2b expression resulted in significant increase of mRNA expression of IL-1β, IL-6, TNF-αand COX-2, as well as obvious reduction of IL-10 in NR8383 cells.
Conclusion
1. A novel gene CNT2b was found and cloned in rat peritoneal macrophages.
2. CNT2b was high-expression gene induced by inflammation, which not significant increase in normal tissues but in macrophages stimulated with inflammation. During the course of acute peritonitis, CNT2b mRNA expression in peritoneal macrophages showed obvious temporal and bimodal characteristics. Anti-inflammatory drugs could decrease the expression level of CNT2b mRNA.
3. Suppression of CNT2b expression resulted in significant increase of pro-inflammatory factor expression and reduction of IL-10 expression.
4. CNT2b involved in regulation process of inflammatory response, and its function was related to suppressing inflammation response.
方法
1.分析不同物种、不同亚型的COX的保守序列,设计简并引物。
2.采用腹腔注射酵母多糖A的方法建立SD大鼠急性腹膜炎模型,采集腹腔巨噬细胞,提取总RNA,进行RT-PCR,扩增产物纯化后连接T载体,转化DH5α细菌,抽提质粒,测序。
3.选取新型序列,进行3’,5’快速末端反应,获取全长基因序列。
4.采用Realtime RT-PCR法分别检测新基因CNT2 mRNA、CNT2b mRNA在大鼠不同脏器组织中的表达情况,以及在酵母多糖诱导的急性腹膜炎大鼠腹腔巨噬细胞中表达的时相特征。
5.应用Realtime RT-PCR法观察抗炎药物阿司匹林(12mg/kg)、塞来昔布(0.13mg/kg)和泼尼松(0.4mg/kg)对大鼠腹腔巨噬细胞中CNT2b mRNA和CNT2 mRNA表达的影响。
6.针对CNT2b序列设计siRNA干扰序列,采用脂质体转染体外培养NR8383大鼠巨噬细胞;Realtime RT-PCR法检测干扰后细胞中炎症相关因子IL-1α、IL-1β、IL-6、TNF-α、COX-2、IL-10以及CNT2、CNT2b等分子的mRNA表达情况。
结果
1.不同物种的COX-1、COX-2和COX-3序列中存在两段氨基酸保守序列。
2.从大鼠腹腔巨噬细胞中发现并克隆了一个新基因,全长13330nt,为一mRNA序列,对应DNA位于大鼠第三号染色体3q35区。经BLAST分析与序列比对,该基因与Na+依赖性核苷转运载体(Na+/nucleoside cotransporter, CNT)2有较高同源性,保留了CNT2的第1、7~14、16、17内含子;并且,在CNT2的最后一个(第18个)外显子后,又附加了一段3169nt长的序列。由于该基因和CNT2均包含加尾信号和polyA尾巴,均为完整的转录产物,故确定为CNT2的剪接异构体,已提交Genbank数据库并获得登录号:EU032627.2,重新命名为CNT2b。
3. CNT2 mRNA在全身各器官均有表达,其中在心脏、肝脏、平滑肌、脾脏表达较高;CNT2b mRNA在正常器官中表达量远远少于CNT2 mRNA,其中心脏、平滑肌、肝脏表达稍高。
4. CNT2b mRNA在腹膜炎大鼠腹腔巨噬细胞中的表达具有显著时相特征。注射酵母多糖后,其表达显著升高,至2h时达到最高点;随后,表达量逐渐降低,至24h时基本恢复至0.5h时的水平;致炎后36h,表达量再次明显升高,至第48h时又降至36h时的47.52%。CNT2 mRNA在腹膜炎大鼠腹腔巨噬细胞中的表达也具有类似特征,其时相变化幅度远少于CNT2b mRNA。
5.三种抗炎药物阿司匹林、泼尼松、塞来昔布均能明显降低CNT2b mRNA表达量。在临床使用剂量下,泼尼松作用最强,阿司匹林次之,塞来昔布最弱。
6.抑制CNT2b基因表达后NR8383细胞中IL-1βmRNA,IL-6 mRNA,TNF-αmRNA,COX-2 mRNA的表达均显著升高,IL-10 mRNA的表达明显降低。
结论
1.在大鼠腹腔巨噬细胞中发现并克隆了一个新基因CNT2b。
2. CNT2b属于炎症诱导性高表达基因,在正常组织中很少表达,而在急性炎症刺激的巨噬细胞中表达显著增加。在大鼠急性腹膜炎过程中,腹腔巨噬细胞CNT2b mRNA的表达具有明显时相性,呈双峰特征。抗炎药物能明显抑制CNT2b mRNA的表达。
3.抑制CNT2b的表达能促进多种促炎因子的表达,降低抗炎因子IL-10的表达。
4. CNT2b参与了炎症反应的调控过程,其功能与抑制炎症反应有关。
Inflammation and immune response is one of the most common pathological processes, and various human diseases are related to it. In addition to well-known infectious diseases, rheumatoid arthritis, rheumatoid disease, systemic lupus erythematosus, liver fibrosis, atherosclerosis, myocardial infarction, Alzheimer's disease, as well as a variety of malignant diseases are closely related to inflammation, which involved in the occurrence, development and prognosis in the whole disease process. Macrophage is an important kind of cell in immune system, which not only has a strong phagocytic function, but also be main antigen-presenting cell, and play a key role in a variety of inflammatory and immune responses. Activated macrophage can secrete a variety of bioactive substances, such as nitric oxide, interleukine-1, tumor necrosis factor-α, reactive oxygen species. Inducible cyclooxygenase (COX), COX-2 can be highly expressed in macrophage. Many studies have suggested that COX-2 is not specific to participate in inflammatory response, but play an important role in protection of gastric mucosa, maintenance of renal function, learning and memory, so other subtypes of COX might exist. A large number of inflammatory factors form the complex inflammation-immune network, which interact, co-regulate and control the process of inflammatory response and prognosis at different stages. To search specific factors for the regulation of inflammatory response will be far-reaching significance to a deep understanding of the pathophysiological mechanisms of inflammation, as well as the treatment of inflammatory diseases.
Methods
1. To design the degenerate primers base on the consensus sequence of different cyclooxytenases isoforms from various species.
2. Acute peritonitis model was established in rats using intraperitoneal injection of zymosan A. Total RNA was extracted from peritoneal macrophages, and then purified PCR products were ligated into T vector and sequenced with automated technology.
3. Full-length sequence of the novel gene was obtained by 3’and 5’rapid amplification of cDNA ends technique.
4. The mRNA expression level of CNT2 and the novel gene CNT2b in different tissues in rats and temporal expression pattern in macrophages from the zymosan A-induced acute peritonitis of rat were detected by Realtime RT-PCR technique.
5. The effects of anti-inflammatory drug aspirin (12mg/kg), celecoxib (0.13mg/kg) and prednisone (0.4mg/kg) on CNT2b mRNA and CNT2 mRNA expression in rat peritoneal macrophages were measured by Realtime RT-PCR.
6. siRNA interference sequence was designed according to CNT2b sequence and transfected into NR8383 rat macrophages using liposomes in vitro.
7. After siRNA interference, mRNA expression of inflammation related factors in NR8383 rat macrophages, such as IL-1α, IL-1β, IL-6, TNF-α, CNT2, CNT2b, COX-2, IL-10 were measured by Realtime RT-PCR.
Results
1. Two conservative amino acid sequences were observed in different species of COX-1, COX-2 and COX-3 sequences.
2. A novel mRNA sequence, 13330nt long, was found and cloned from rat peritoneal macrophages, which corresponding DNA is located at rat chromosome 3q35. BLAST analysis and sequence alignment indicated that the novel gene had high homology with Na+-dependent nucleoside transporters (Na+/nucleoside cotransporter, CNT) 2, which retained the intron 1,7-14,16,17 and a 3197nt sequences was appendixed to the last exon of CNT2. Since the novel gene and CNT2 were both complete transcriptions, which contained tailing signal and poly A tail, it was identified as the splicing isomers of CNT2 and named as CNT2b.
3. CNT2 mRNA was expressed in different tissues and the level was higher in heart, liver, skeletal muscle and spleen; CNT2b mRNA expression level in normal organs was far less than that of CNT2, and the level was slightly higher in heart, skeletal muscle and liver.
4. The significant phase characteristics of CNT2b mRNA expression was observed in peritoneal macrophages from peritonitis rat. After zymosan injection, CNT2b mRNA expression level was significantly increased and reached the peak point at 2h; subsequently, the expression level was gradually decreased, and returned to their levels at 0.5h until 24h. At 36th hour after inflammatory stimulation, CNT2b mRNA expression level was significantly increased again. The expression level of CNT2b mRNA was decreased by about 53% at 48th hour after inflammatory stimulation by comparison of that at 36th hour.
5. After treated with aspirin, prednisone and celecoxib, respectively, CNT2b mRNA expression decreased significantly, in which the effect of prednisone was strongest and celecoxib weakest.
6. Suppression of CNT2b expression resulted in significant increase of mRNA expression of IL-1β, IL-6, TNF-αand COX-2, as well as obvious reduction of IL-10 in NR8383 cells.
Conclusion
1. A novel gene CNT2b was found and cloned in rat peritoneal macrophages.
2. CNT2b was high-expression gene induced by inflammation, which not significant increase in normal tissues but in macrophages stimulated with inflammation. During the course of acute peritonitis, CNT2b mRNA expression in peritoneal macrophages showed obvious temporal and bimodal characteristics. Anti-inflammatory drugs could decrease the expression level of CNT2b mRNA.
3. Suppression of CNT2b expression resulted in significant increase of pro-inflammatory factor expression and reduction of IL-10 expression.
4. CNT2b involved in regulation process of inflammatory response, and its function was related to suppressing inflammation response.
引文
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