本文選題：SET　切入點：RNA干擾　出處：《湖南師范大學》2010年碩士論文　論文類型：學位論文

【摘要】：目的：初步闡明SET蛋白在三氯乙烯(TCE)致肝細胞毒性中的作用,為探討SET蛋白作為TCE職業(yè)中毒效應生物標志物的可能性及揭示TCE肝細胞毒性的可能機制提供依據(jù)。 方法：在課題組前期初步篩選的TCE差異蛋白基礎上,選取具有重要生物學功能的差異蛋白——蛋白磷酸酶2A(PP2A)抑制蛋白SET,采用慢病毒介導的RNAi技術構建穩(wěn)定干擾SET肝細胞株；不同濃度(0.25 mmol/L、0.5 mmol/L、1 mmol/L、2mmol/L、4 mmol/L、8 mmol/L) TCE對L-02肝細胞進行不同時間點(6 h、12 h、24 h)處理后,利用Western Blot、實時熒光定量PCR、PP2A活性試劑盒檢測不同濃度、不同時間點TCE處理L-02肝細胞后SET的表達,并探討SET表達改變與TCE刺激肝細胞后細胞形態(tài)、細胞增殖、細胞凋亡以及SET蛋白亞細胞定位改變等指標的相互關系。 結果：①測序鑒定結果表明,靶向SET的shRNA慢病毒表達載體構建成功。Western Blot和實時熒光定量PCR檢測顯示,psiRNA4組肝細胞干擾效果最好且穩(wěn)定,對SET表達的抑制率達到90%以上。②用5、10、15、20、25、30、35、40 mmol/L TCE分別處理L-02肝細胞24 h后,CCK-8法顯示TCE可顯著抑制L-02肝細胞的增殖活性,呈明顯濃度-效應關系,其對肝細胞的半數(shù)抑制濃度(IC50)為15.95 mmol/L。③Western Blot和實時熒光定量PCR結果顯示,不同濃度、不同時間點TCE處理后肝細胞內SET mRNA、SET蛋白的表達均上升,其中TCE處理12 h效應最顯著。而PP2A活性檢測顯示,不同濃度、不同時間點TCE處理后肝細胞內PP2A活性下降,進一步驗證了Western Blot、實時熒光定量PCR的結果。④不同濃度(0.25、1.0、4.0 mmol/L) TCE分別處理正常肝細胞和穩(wěn)定干擾SET肝細胞后,相同濃度TCE處理的正常肝細胞組和穩(wěn)定干擾SET肝細胞組的細胞凋亡比較結果顯示,SET表達抑制后可促進肝細胞凋亡。同時,穩(wěn)定干擾SET肝細胞各組隨著TCE濃度的增加,細胞凋亡率也呈現(xiàn)一定的上升趨勢。⑤SET表達抑制后肝細胞增殖速度減慢,形態(tài)變小,邊緣模糊。⑥SET蛋白表達于肝細胞核內,不同濃度TCE處理肝細胞后未導致SET蛋白的亞細胞定位發(fā)生改變。 結論：①成功構建針對SET的慢病毒干擾載體,篩選出穩(wěn)定干擾SET肝細胞株。②TCE對肝細胞具有細胞毒性,TCE處理24 h對肝細胞的半數(shù)抑制濃度為15.95 mmol/L。③TCE處理肝細胞后可誘導SET表達增加。④SET被抑制表達后肝細胞增殖活性下降。⑤SET蛋白可抑制肝細胞凋亡,TCE可促進細胞凋亡。⑥SET蛋白表達于肝細胞核內,TCE處理肝細胞后不會導致SET蛋白的亞細胞定位發(fā)生改變。
[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.
【學位授予單位】：湖南師范大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R181.3

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本文編號：1597609