RNAi沉默线粒体氯通道表达在过氧化氢诱导的大鼠胶质细胞损伤过程中作用与机制的研究
发布时间：2019-01-10 07:30
【摘要】： 目的:探讨线粒体氯通道/细胞内氯通道4(mtCLIC/CLIC4)在H_2O_2诱导的大鼠胶质细胞损伤过程中作用及机制。 方法:根据RNAi设计原理以及基因重组技术构建siRNA-CLIC4表达载体,转染SD大鼠C6胶质瘤细胞,对比观察CLIC4和CLIC4-RNAi在H_2O_2诱导的大鼠胶质细胞损伤过程中的作用。 结果:(1)H_2O_2诱导的大鼠C6细胞损伤过程中,CLIC4蛋白表达显著增强,同时Caspase3、VDAC1蛋白表达明显增强以及Bax/Bcl-2比值明显增高。(2)首次完成大鼠C6胶质瘤细胞CLIC4cDNA钓取,亚克隆和测序,发现新的SD大鼠胶质细胞CLIC4序列,将结果登录到GenBank,获得登录号:Bankit878161,EF397567。(3)首次利用RNAi技术成功构建siRNA-CLIC4表达质粒,显著抑制CLIC4基因表达,并获得稳定转染C6细胞株。(4)在CLIC4干涉组(pSH1Si-CLIC4)中尽管总CLIC4蛋白表达在RNAi作用下明显下调,但伴随H_2O_2浓度的增加,细胞核内的CLIC4蛋白表达仍显著增加,促进了H_2O_2诱导的细胞凋亡。 结论:发现新的SD大鼠胶质细胞CLIC4cDNA的基因序列并成功构建siRNA-CLIC4有效表达质粒及稳定转染的C6细胞株。在H_2O_2诱导的C6细胞损伤过程中,CLIC4作为凋亡效应分子参与了其线粒体途径的凋亡过程。而通过RNAi技术下调CLIC4表达后,同样明显促进了H_2O_2诱导的细胞凋亡,该过程中细胞核靶向的CLIC4大量入核,可能与CLIC4作为离子通道影响了细胞核内离子平衡并改变其pH值水平有关,进而通过pH-依赖的核酸内切酶激活的细胞核凋亡途径加速细胞凋亡。 本研究为探讨细胞内氯通道在神经退行性疾病发病过程中所发挥的作用进行了新的探索,也为寻找肿瘤基因治疗新的作用靶点进行了有益的尝试。
[Abstract]:Aim: to investigate the role and mechanism of mitochondrial chloride channel / intracellular chloride channel 4 (mtCLIC/CLIC4) in H_2O_2 induced glial injury in rats. Methods: according to the principle of RNAi design and gene recombination, the siRNA-CLIC4 expression vector was constructed and transfected into SD rat C6 glioma cells. The role of CLIC4 and CLIC4-RNAi in the process of H_2O_2 induced glial cell injury was observed. Results: (1) during the injury of C6 cells induced by H_2O_2, the expression of CLIC4 protein was significantly increased, and the expression of Caspase3, was also significantly increased. The expression of VDAC1 protein and the ratio of Bax/Bcl-2 were significantly increased. (2) A novel CLIC4 sequence of rat glial cells of SD was found after CLIC4cDNA fishing, subcloning and sequencing of rat C6 glioma cells were completed for the first time, and the results were logged into GenBank,. Accession number: Bankit878161,EF397567. (3) successfully constructed siRNA-CLIC4 expression plasmid by RNAi technique for the first time, and significantly inhibited the expression of CLIC4 gene. Stable transfection of C6 cell line was obtained. (4) in CLIC4 interference group (pSH1Si-CLIC4), although the expression of total CLIC4 protein was significantly down-regulated by RNAi, the expression of CLIC4 protein in the nucleus was still significantly increased with the increase of H_2O_2 concentration. H_2O_2-induced apoptosis was promoted. Conclusion: the new gene sequence of CLIC4cDNA in glial cells of SD rats was found and the effective expression plasmid of siRNA-CLIC4 and the stable transfected C6 cell line were successfully constructed. In the process of C6 cell injury induced by H_2O_2, CLIC4 is involved in the apoptosis process of mitochondria pathway as an effector of apoptosis. After down-regulation of CLIC4 expression by RNAi technique, apoptosis induced by H_2O_2 was also promoted, during which a large number of nuclear targeted CLIC4 entered the nucleus. It may be related to the effect of CLIC4 as an ion channel on the ion balance in the nucleus and to change the level of pH, and then accelerate the apoptosis through the pathway of nucleus apoptosis activated by pH- dependent endonuclease. In order to explore the role of intracellular chloride channels in the pathogenesis of neurodegenerative diseases, we have made a new attempt to find new targets for tumor gene therapy.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363
本文编号：2406053
[Abstract]:Aim: to investigate the role and mechanism of mitochondrial chloride channel / intracellular chloride channel 4 (mtCLIC/CLIC4) in H_2O_2 induced glial injury in rats. Methods: according to the principle of RNAi design and gene recombination, the siRNA-CLIC4 expression vector was constructed and transfected into SD rat C6 glioma cells. The role of CLIC4 and CLIC4-RNAi in the process of H_2O_2 induced glial cell injury was observed. Results: (1) during the injury of C6 cells induced by H_2O_2, the expression of CLIC4 protein was significantly increased, and the expression of Caspase3, was also significantly increased. The expression of VDAC1 protein and the ratio of Bax/Bcl-2 were significantly increased. (2) A novel CLIC4 sequence of rat glial cells of SD was found after CLIC4cDNA fishing, subcloning and sequencing of rat C6 glioma cells were completed for the first time, and the results were logged into GenBank,. Accession number: Bankit878161,EF397567. (3) successfully constructed siRNA-CLIC4 expression plasmid by RNAi technique for the first time, and significantly inhibited the expression of CLIC4 gene. Stable transfection of C6 cell line was obtained. (4) in CLIC4 interference group (pSH1Si-CLIC4), although the expression of total CLIC4 protein was significantly down-regulated by RNAi, the expression of CLIC4 protein in the nucleus was still significantly increased with the increase of H_2O_2 concentration. H_2O_2-induced apoptosis was promoted. Conclusion: the new gene sequence of CLIC4cDNA in glial cells of SD rats was found and the effective expression plasmid of siRNA-CLIC4 and the stable transfected C6 cell line were successfully constructed. In the process of C6 cell injury induced by H_2O_2, CLIC4 is involved in the apoptosis process of mitochondria pathway as an effector of apoptosis. After down-regulation of CLIC4 expression by RNAi technique, apoptosis induced by H_2O_2 was also promoted, during which a large number of nuclear targeted CLIC4 entered the nucleus. It may be related to the effect of CLIC4 as an ion channel on the ion balance in the nucleus and to change the level of pH, and then accelerate the apoptosis through the pathway of nucleus apoptosis activated by pH- dependent endonuclease. In order to explore the role of intracellular chloride channels in the pathogenesis of neurodegenerative diseases, we have made a new attempt to find new targets for tumor gene therapy.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363
【引证文献】
相关博士学位论文 前1条
1 张海宁;血管性认知障碍大鼠脑突触小体的差异蛋白质组学研究[D];吉林大学;2011年
，本文编号：2406053
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