本文選題：胱硫醚-γ-裂解酶 + 內(nèi)源性硫化氫��； 參考：《河南大學(xué)》2012年碩士論文

【摘要】：目的：研究內(nèi)源性CSE/H_2S系統(tǒng)對細胞氧化損傷的保護作用，并初步探討其作用機制。 方法：胱硫醚-γ-裂解酶（cystathionine-γ-lyase，，CSE）特異性siRNA轉(zhuǎn)染HepG2、HEK293和IMR90細胞，CSE抑制劑PAG分別作用于不同細胞，MTS法檢測H_2O_2對細胞的損傷效果；熒光探針法檢測細胞中內(nèi)超氧陰離子（superoxide anion，O~(2-)）和活性氧自由基（reactive oxygen species，ROS）的含量；GSSH/GSH試劑盒分析細胞內(nèi)還原型谷胱甘肽（glutathione，GSH）的水平。重組質(zhì)粒pEGFP-CSE轉(zhuǎn)染HEK293細胞，分別檢測細胞中O~(2-)、ROS和GSH的水平。Western blot法檢測CSE-siRNA轉(zhuǎn)染細胞中轉(zhuǎn)錄因子Nrf2（nuclear factor erythroid2p45related factor2）的蛋白表達，初步探討其作用機制。 結(jié)果：H_2O_2可誘導(dǎo)HepG2、HEK293和IMR90細胞損傷，作用12h后，IC50值分別為500±11μM（HepG2），200±39μM（HEK293），202±19μM（IMR90）。CSE-siRNA和PAG作用于細胞后，H_2O_2對細胞的損傷作用明顯增加，細胞中O~(2-)、ROS含量顯著增加，GSH水平明顯降低。重組質(zhì)粒pEGFP-CSE轉(zhuǎn)染HEK293細胞后，細胞中的O~(2-)、ROS含量明顯減低，GSH水平顯著增加。Western blot結(jié)果顯示CSE-siRNA轉(zhuǎn)染HepG2、HEK293細胞后，Nrf2的表達無明顯變化。 結(jié)論： 1.抑制細胞中CSE的表達可增強H_2O_2誘導(dǎo)的細胞損傷。 2．抑制細胞中CSE的表達，可使細胞內(nèi)超氧離子（O~(2-)）和活性氧自由基（ROS）含量增加，還原型谷胱甘肽（GSH）水平降低；以CSE重組質(zhì)粒轉(zhuǎn)染HEK293細胞，可通過提高細胞中CSE的表達，來降低細胞中O~(2-)和ROS的含量，提高GSH水平；表明內(nèi)源性CSE/H_2S系統(tǒng)對細胞氧化損傷具有保護作用。 3．CSE/H_2S對細胞中Nrf2蛋白的表達無明顯影響。
[Abstract]:Aim: to study the protective effect of endogenous CSE/H_2S system on cell oxidative injury and its mechanism. Methods: cystathionine- 緯 -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 鹵11 渭 MHEK293N, 202 鹵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.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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