本文選題：肝癌細(xì)胞株 + RASSF1A基因甲基化��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的探討RASSF1A基因甲基化在肝癌細(xì)胞株中表達(dá)情況及5-Aza-Cd R對人肝癌細(xì)胞株Hep G2細(xì)胞增殖及RASSF1A基因m RNA表達(dá)的影響。方法體外培養(yǎng)肝癌細(xì)胞株及正常肝細(xì)胞株,應(yīng)用MSP法分別檢測3種肝癌細(xì)胞株Hep G2、Hep3B、SK-HEP1及正常肝細(xì)胞株LO2 RASSF1A甲基化水平,以探討RASSF1A甲基化在肝癌細(xì)胞株的表達(dá)情況。以肝癌細(xì)胞株Hep G2為研究對象,實(shí)驗(yàn)組分別以不同濃度的甲基化抑制劑5-Aza-Cd R(0.5μmol/L、5μmol/L、50μmol/L)對人肝癌細(xì)胞株Hep G2去甲基化組處理,對照組5-Aza-Cd R濃度為0μmol/L;采用CCK-8法檢測兩組肝癌細(xì)胞增殖情況的變化,以觀察甲基化抑制劑對肝癌細(xì)胞增值的影響,并采用RT-PCR檢測5-Aza-Cd R處理前、后Hep G2細(xì)胞抑癌基因RASSF1A基因m RNA表達(dá)水平的變化。結(jié)果人肝癌細(xì)胞株Hep G2、Hep3B、SK-HEP1均檢測到RASSF1A基因甲基化表達(dá),而正常肝細(xì)胞株LO2中未檢測到RASSF1A基因的甲基化表達(dá),提示肝細(xì)胞癌變與RASSF1A基因甲基化有關(guān)。5-Aza-Cd R可以抑制肝癌細(xì)胞Hep G2的生長增值。處理72小時(shí)后,抑制率最明顯,且隨著藥物濃度增加,增殖抑制率越明顯,表明去甲基化可以抑制細(xì)胞生長增值(F=44.164,P0.01)。5-Aza-Cd R處理后實(shí)驗(yàn)組較對照組RASSF1A基因m RNA表達(dá)明顯上調(diào),呈劑量依賴性。在50μmol/L時(shí)去甲基化效果最明顯。50μmol/L 5-Aza-Cd R處理24h、48h、72h時(shí)RASSF1A基因m RNA相對表達(dá)量分別為(0.2798±0.0048、0.3678±0.0161、0.3873±0.00105),與對照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),表明去甲基化后可以增加Hep G2細(xì)胞RASSF1A基因m RNA表達(dá)。結(jié)論1.肝細(xì)胞癌變與抑癌基因RASSF1A甲基化有關(guān)。2.5-Aza-CdR可以抑制HepG2細(xì)胞生長增殖,呈劑量依賴性,其機(jī)制可能與其調(diào)控RASSF1A基因啟動子甲基化水平,從而使RASSF1A基因m RNA表達(dá)上調(diào)有關(guān)。
[Abstract]:Objective to investigate the expression of RASSF1A gene methylation in human hepatoma cell line and the effect of 5-Aza-CD R on proliferation and mRNA expression of RASSF1A gene in human hepatoma cell line Hep G2. Methods the methylation level of RASSF1A methylation in three hepatoma cell lines Hep G2H2H2Bh3BP1and normal hepatocyte line LO2 RASSF1A was detected by MSP method in order to investigate the expression of RASSF1A methylation in hepatoma cells. Human hepatoma cell line HepG2 was treated with methylation inhibitor 5-Aza-CD R 0.5 渭 mol / L 5 渭 mol 路L ~ (-1) / L ~ (50 渭 mol 路L ~ (-1), respectively. In the control group, the concentration of 5-Aza-CD R was 0 渭 mol / L, the proliferation of hepatoma cells was detected by CCK-8 method, and the effect of methylation inhibitor on the proliferation of hepatoma cells was observed, and the effect of 5-Aza-CD R on the proliferation of hepatoma cells was detected by RT-PCR. Changes of mRNA expression of tumor suppressor gene RASSF1A in Hep G2 cells. Results RASSF1A gene methylation was detected in human hepatoma cell line Hep G _ 2H _ 3B _ (3) B _ (+) -HEP1, but no methylation expression of RASSF1A gene was detected in normal hepatoma cell line LO2, and the methylation of RASSF1A gene was not detected in normal hepatoma cell line (LO2). The results suggest that the carcinogenesis of hepatocytes is related to the methylation of RASSF1A gene. 5-Aza-CD R can inhibit the proliferation of HepG2 cells. After 72 hours of treatment, the inhibition rate was the most obvious, and with the increase of drug concentration, the inhibition rate of proliferation was more obvious, which indicated that demethylation could inhibit the growth and proliferation of the cells. The mRNA expression of RASSF1A gene in the experimental group was significantly up-regulated than that in the control group. In a dose-dependent manner. The relative mRNA expression of RASSF1A gene was 0.2798 鹵0.0048 鹵0.3678 鹵0.0161 鹵0.3873 鹵0.00105 after treatment with 50 渭 mol / L 5-Aza-CD R for 24 h and 48 h / 72 h, respectively, which was significantly different from that of the control group (P 0.05), indicating that the mRNA expression of RASSF1A gene in Hep G2 cells could be increased after demethylation. Conclusion 1. The carcinogenesis of hepatocytes is related to the methylation of tumor suppressor gene RASSF1A. 2.5-Aza-CdR can inhibit the growth and proliferation of HepG2 cells in a dose-dependent manner. The mechanism may be related to its regulation of methylation level of RASSF1A gene promoter and up-regulation of mRNA expression of RASSF1A gene.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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本文編號：2017594