發(fā)布時(shí)間：2018-03-11 09:39

  本文選題：SET　切入點(diǎn)：RNA干擾　出處：《湖南師范大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：初步闡明SET蛋白在三氯乙烯(TCE)致肝細(xì)胞毒性中的作用,為探討SET蛋白作為T(mén)CE職業(yè)中毒效應(yīng)生物標(biāo)志物的可能性及揭示TCE肝細(xì)胞毒性的可能機(jī)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(chǔ)上,選取具有重要生物學(xué)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(dǎo)的RNAi技術(shù)構(gòu)建穩(wěn)定干擾SET肝細(xì)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(duì)L-02肝細(xì)胞進(jìn)行不同時(shí)間點(diǎn)(6 h、12 h、24 h)處理后,利用Western Blot、實(shí)時(shí)熒光定量PCR、PP2A活性試劑盒檢測(cè)不同濃度、不同時(shí)間點(diǎn)TCE處理L-02肝細(xì)胞后SET的表達(dá),并探討SET表達(dá)改變與TCE刺激肝細(xì)胞后細(xì)胞形態(tài)、細(xì)胞增殖、細(xì)胞凋亡以及SET蛋白亞細(xì)胞定位改變等指標(biāo)的相互關(guān)系。 結(jié)果：①測(cè)序鑒定結(jié)果表明,靶向SET的shRNA慢病毒表達(dá)載體構(gòu)建成功。Western Blot和實(shí)時(shí)熒光定量PCR檢測(cè)顯示,psiRNA4組肝細(xì)胞干擾效果最好且穩(wěn)定,對(duì)SET表達(dá)的抑制率達(dá)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(xì)胞24 h后,CCK-8法顯示TCE可顯著抑制L-02肝細(xì)胞的增殖活性,呈明顯濃度-效應(yīng)關(guān)系,其對(duì)肝細(xì)胞的半數(shù)抑制濃度(IC50)為15.95 mmol/L。③Western Blot和實(shí)時(shí)熒光定量PCR結(jié)果顯示,不同濃度、不同時(shí)間點(diǎn)TCE處理后肝細(xì)胞內(nèi)SET mRNA、SET蛋白的表達(dá)均上升,其中TCE處理12 h效應(yīng)最顯著。而PP2A活性檢測(cè)顯示,不同濃度、不同時(shí)間點(diǎn)TCE處理后肝細(xì)胞內(nèi)PP2A活性下降,進(jìn)一步驗(yàn)證了Western Blot、實(shí)時(shí)熒光定量PCR的結(jié)果。④不同濃度(0.25、1.0、4.0 mmol/L) TCE分別處理正常肝細(xì)胞和穩(wěn)定干擾SET肝細(xì)胞后,相同濃度TCE處理的正常肝細(xì)胞組和穩(wěn)定干擾SET肝細(xì)胞組的細(xì)胞凋亡比較結(jié)果顯示,SET表達(dá)抑制后可促進(jìn)肝細(xì)胞凋亡。同時(shí),穩(wěn)定干擾SET肝細(xì)胞各組隨著TCE濃度的增加,細(xì)胞凋亡率也呈現(xiàn)一定的上升趨勢(shì)。⑤SET表達(dá)抑制后肝細(xì)胞增殖速度減慢,形態(tài)變小,邊緣模糊。⑥SET蛋白表達(dá)于肝細(xì)胞核內(nèi),不同濃度TCE處理肝細(xì)胞后未導(dǎo)致SET蛋白的亞細(xì)胞定位發(fā)生改變。 結(jié)論：①成功構(gòu)建針對(duì)SET的慢病毒干擾載體,篩選出穩(wěn)定干擾SET肝細(xì)胞株。②TCE對(duì)肝細(xì)胞具有細(xì)胞毒性,TCE處理24 h對(duì)肝細(xì)胞的半數(shù)抑制濃度為15.95 mmol/L。③TCE處理肝細(xì)胞后可誘導(dǎo)SET表達(dá)增加。④SET被抑制表達(dá)后肝細(xì)胞增殖活性下降。⑤SET蛋白可抑制肝細(xì)胞凋亡,TCE可促進(jìn)細(xì)胞凋亡。⑥SET蛋白表達(dá)于肝細(xì)胞核內(nèi),TCE處理肝細(xì)胞后不會(huì)導(dǎo)致SET蛋白的亞細(xì)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(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R181.3

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本文編號(hào)：1597609