SET在三氯乙烯肝细胞毒性中的作用及其流行病学意义

发布时间：2018-03-11 09:39

本文选题：SET　切入点：RNA干扰　出处：《湖南师范大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的：初步阐明SET蛋白在三氯乙烯(TCE)致肝细胞毒性中的作用,为探讨SET蛋白作为TCE职业中毒效应生物标志物的可能性及揭示TCE肝细胞毒性的可能机制提供依据。方法：在课题组前期初步筛选的TCE差异蛋白基础上,选取具有重要生物学功能的差异蛋白——蛋白磷酸酶2A(PP2A)抑制蛋白SET,采用慢病毒介导的RNAi技术构建稳定干扰SET肝细胞株；不同浓度(0.25 mmol/L、0.5 mmol/L、1 mmol/L、2mmol/L、4 mmol/L、8 mmol/L) TCE对L-02肝细胞进行不同时间点(6 h、12 h、24 h)处理后,利用Western Blot、实时荧光定量PCR、PP2A活性试剂盒检测不同浓度、不同时间点TCE处理L-02肝细胞后SET的表达,并探讨SET表达改变与TCE刺激肝细胞后细胞形态、细胞增殖、细胞凋亡以及SET蛋白亚细胞定位改变等指标的相互关系。结果：①测序鉴定结果表明,靶向SET的shRNA慢病毒表达载体构建成功。Western Blot和实时荧光定量PCR检测显示,psiRNA4组肝细胞干扰效果最好且稳定,对SET表达的抑制率达到90%以上。②用5、10、15、20、25、30、35、40 mmol/L TCE分别处理L-02肝细胞24 h后,CCK-8法显示TCE可显著抑制L-02肝细胞的增殖活性,呈明显浓度-效应关系,其对肝细胞的半数抑制浓度(IC50)为15.95 mmol/L。③Western Blot和实时荧光定量PCR结果显示,不同浓度、不同时间点TCE处理后肝细胞内SET mRNA、SET蛋白的表达均上升,其中TCE处理12 h效应最显著。而PP2A活性检测显示,不同浓度、不同时间点TCE处理后肝细胞内PP2A活性下降,进一步验证了Western Blot、实时荧光定量PCR的结果。④不同浓度(0.25、1.0、4.0 mmol/L) TCE分别处理正常肝细胞和稳定干扰SET肝细胞后,相同浓度TCE处理的正常肝细胞组和稳定干扰SET肝细胞组的细胞凋亡比较结果显示,SET表达抑制后可促进肝细胞凋亡。同时,稳定干扰SET肝细胞各组随着TCE浓度的增加,细胞凋亡率也呈现一定的上升趋势。⑤SET表达抑制后肝细胞增殖速度减慢,形态变小,边缘模糊。⑥SET蛋白表达于肝细胞核内,不同浓度TCE处理肝细胞后未导致SET蛋白的亚细胞定位发生改变。结论：①成功构建针对SET的慢病毒干扰载体,筛选出稳定干扰SET肝细胞株。②TCE对肝细胞具有细胞毒性,TCE处理24 h对肝细胞的半数抑制浓度为15.95 mmol/L。③TCE处理肝细胞后可诱导SET表达增加。④SET被抑制表达后肝细胞增殖活性下降。⑤SET蛋白可抑制肝细胞凋亡,TCE可促进细胞凋亡。⑥SET蛋白表达于肝细胞核内,TCE处理肝细胞后不会导致SET蛋白的亚细胞定位发生改变。
[Abstract]:Objective: to clarify the role of SET protein in hepatocytotoxicity induced by trichloroethylene (TCE), and to provide a basis for exploring the possibility of SET protein as a biomarker of TCE occupational toxicity and revealing the possible mechanism of TCE hepatocytotoxicity. Methods: based on the preliminary screening of TCE differential protein in the early stage of the study group, we selected the differential protein-protein PP2A inhibitory protein (set), which has important biological function, and constructed the stable interfering SET hepatocyte cell line by lentivirus mediated RNAi technique. L-02 hepatocytes were treated with different concentrations of 0.25 mmol / L 0.5 mmol / L 0.5 mmol / L 1 mmol / L 1 mmol / L 2 mmol / L 2 mmol / L and 4 mmol / L 4 mmol / L TCE at different time points (6 h / 12 h / 24 h). The expression of SET in L-02 hepatocytes was detected by Western blot, real-time fluorescence quantitative PCRPP2A activity kit, and TCE treatment at different time points after L-02 hepatocytes were treated with TCE. The relationship between the expression of SET and the changes of cell morphology, cell proliferation, apoptosis and subcellular localization of SET protein after TCE stimulation was investigated. Results the shRNA lentivirus expression vector targeting SET was successfully constructed. Western Blot and real-time fluorescence quantitative PCR analysis showed that the interference effect of psiRNA4 group was the best and stable. The inhibitory rate of SET expression was more than 90%. After the L-02 hepatocytes were treated with 5A10101520A2530A3540 mmol/L TCE for 24 h, CCK-8 method showed that TCE could significantly inhibit the proliferation of L-02 hepatocytes in a dose-dependent manner. IC50 was 15.95 mmol/L.3Western Blot and real-time quantitative PCR showed that the expression of SET mRNA-set protein in hepatocytes was increased after TCE treatment at different concentrations and at different time points. The effect of TCE treatment for 12 h was the most significant, while the activity of PP2A decreased after TCE treatment at different concentrations and at different time points. It was further verified that Western blot.4 different concentrations of 0.251.0 渭 mol / L TCE were used to treat normal hepatocytes and stable interfering SET hepatocytes, respectively, as a result of real-time fluorescence quantitative PCR, 4. 4 mmol / L TCE. The comparison of apoptosis between the normal hepatocytes treated with the same concentration of TCE and the stable interfering SET hepatocytes showed that the inhibition of the expression of set could promote the apoptosis of hepatocytes. Meanwhile, the stable interference of SET hepatocytes increased with the increase of TCE concentration. The apoptotic rate also showed an upward trend. The proliferation rate of hepatocytes decreased after inhibition of 5SET expression, and the morphology of hepatocytes became smaller, and the blurry edge of .6SET protein was expressed in the hepatocyte nucleus. The subcellular localization of SET protein was not changed after treatment with different concentrations of TCE. Conclusion the lentivirus interference vector targeting SET was successfully constructed by using 1: 1. A stable interfering SET hepatocyte line. 2TCE was found to be cytotoxic to hepatocytes. The 50% inhibitory concentration of TCE on hepatocytes after 24 h treatment was 15.95 mmol/L.3TCE, which could induce the increase of SET expression. 4SET was inhibited and hepatocyte proliferative activity was induced. The decrease of .5SET protein can inhibit the apoptosis of hepatocytes. TCE-6SET protein can promote the expression of apoptosis-induced subcellular localization of SET protein in hepatocytes treated with TCE-treated hepatocytes.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R181.3

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