内源性硫化氢对细胞氧化损伤的保护及机制研究

发布时间：2018-05-31 01:00

本文选题：胱硫醚-γ-裂解酶 + 内源性硫化氢　；参考：《河南大学》2012年硕士论文

【摘要】：目的：研究内源性CSE/H_2S系统对细胞氧化损伤的保护作用，并初步探讨其作用机制。方法：胱硫醚-γ-裂解酶（cystathionine-γ-lyase，，CSE）特异性siRNA转染HepG2、HEK293和IMR90细胞，CSE抑制剂PAG分别作用于不同细胞，MTS法检测H_2O_2对细胞的损伤效果；荧光探针法检测细胞中内超氧阴离子（superoxide anion，O~(2-)）和活性氧自由基（reactive oxygen species，ROS）的含量；GSSH/GSH试剂盒分析细胞内还原型谷胱甘肽（glutathione，GSH）的水平。重组质粒pEGFP-CSE转染HEK293细胞，分别检测细胞中O~(2-)、ROS和GSH的水平。Western blot法检测CSE-siRNA转染细胞中转录因子Nrf2（nuclear factor erythroid2p45related factor2）的蛋白表达，初步探讨其作用机制。结果：H_2O_2可诱导HepG2、HEK293和IMR90细胞损伤，作用12h后，IC50值分别为500±11μM（HepG2），200±39μM（HEK293），202±19μM（IMR90）。CSE-siRNA和PAG作用于细胞后，H_2O_2对细胞的损伤作用明显增加，细胞中O~(2-)、ROS含量显著增加，GSH水平明显降低。重组质粒pEGFP-CSE转染HEK293细胞后，细胞中的O~(2-)、ROS含量明显减低，GSH水平显著增加。Western blot结果显示CSE-siRNA转染HepG2、HEK293细胞后，Nrf2的表达无明显变化。结论： 1.抑制细胞中CSE的表达可增强H_2O_2诱导的细胞损伤。 2．抑制细胞中CSE的表达，可使细胞内超氧离子（O~(2-)）和活性氧自由基（ROS）含量增加，还原型谷胱甘肽（GSH）水平降低；以CSE重组质粒转染HEK293细胞，可通过提高细胞中CSE的表达，来降低细胞中O~(2-)和ROS的含量，提高GSH水平；表明内源性CSE/H_2S系统对细胞氧化损伤具有保护作用。 3．CSE/H_2S对细胞中Nrf2蛋白的表达无明显影响。
[Abstract]:Aim: to study the protective effect of endogenous CSE/H_2S system on cell oxidative injury and its mechanism. Methods: cystathionine- 纬 -lyase specific siRNA was transfected into HepG2pHEK293 and IMR90 cell line PAG to detect the damage effect of H_2O_2 on different cells. The content of superoxide anion (superoxide anions) and reactive oxygen (oxygen) in the cells were detected by fluorescence probe method. The GSSH / GSH kit was used to analyze the level of glutathione (GSH) in the cells. HEK293 cells were transfected with recombinant plasmid pEGFP-CSE. The protein expression of transcription factor Nrf2(nuclear factor erythroid2p45related factor2 (Nrf2(nuclear factor erythroid2p45related factor2) in CSE-siRNA transfected cells was detected by Western blot. Results the injury of HepG2HEK293 and IMR90 cells was induced by: 1 / H2O2. The IC50 of HepG2H2O2 was 500 卤11 渭 MHEK293N, 202 卤19 渭 M(IMR90).CSE-siRNA and PAG, respectively. After 12 hours of exposure, the damage of HepG2HEK293 + 19 渭 M(IMR90).CSE-siRNA and PAG was significantly increased, and the level of GSH was significantly decreased. After the recombinant plasmid pEGFP-CSE was transfected into HEK293 cells, the content of Oligonidin 2 was significantly decreased. The results of Western blot showed that the expression of nrf2 in HepG2 + HEK293 cells was not changed after CSE-siRNA transfection. Conclusion: 1. Inhibiting the expression of CSE in cells can enhance the damage induced by H_2O_2. 2. Inhibiting the expression of CSE could increase the content of superoxide ion (CSE) and reactive oxygen free radical (Ros), and decrease the level of reduced glutathione (GSH). Transfection of HEK293 cells with CSE recombinant plasmid could increase the expression of CSE. The results showed that endogenous CSE/H_2S system had protective effect on oxidative damage of the cells by decreasing the content of OF2-and ROS and increasing the level of GSH. 3.CSE/H_2S had no effect on the expression of Nrf2 protein.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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