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目的研究赖氨大黄酸在体外对乙型肝炎病毒(HBV)增殖及病毒抗原表达的影响及分子机制,为研究赖氨大黄酸抗HBV作用机制奠定初步的实验依据。方法将HepG2.2.15细胞分为实验组(分别加入5、10、20、40、80μmol/L赖氨大黄酸)及不加赖氨大黄酸的对照组,分别培养24、48h收获上清液,采用四唑氮化合物(MTS)法检测细胞的增殖活性,采用ELISA法检测HBsAg和HBeAg的表达水平,实时定量PCR(RT-PCT)检测其中HBV DNA的拷贝数及miR-210、miR-185、miR-199a-3p、miR-370、miR-196a、miR-184、miR-217的表达水平。miR-210、miR-185、miR-199a-3p、miR-370、miR-196a、miR-184、miR-217反义链(ASO)分别转染HepG2.2.15细胞后,MTS法检测细胞的增殖活性,ELISA法检测HBsAg和HBeAg的表达水平。结果在没有对HepG2.2.15细胞的增殖活性产生影响的情况下,赖氨大黄酸抑制了HBsAg的产生和HBV的增殖。20μmol/L的赖氨大黄酸明显促进了miR-210、miR-185、miR-199a-3p的表达,而抑制了miR-370的表达,与对照组相比较,差异具有统计学意义(P<0.05)。结论赖氨大黄酸可能通过影响细胞内miRNA的表达抑制HepG2.2.15细胞中HBV的增殖和HBsAg的产生。
Objective To study the effect and molecular mechanism of lysine on the proliferation and viral antigen expression of hepatitis B virus (HBV) in vitro and lay a preliminary experimental basis for studying the anti-HBV mechanism of lysine. Methods HepG2.2.15 cells were divided into experimental group (5, 10, 20, 40, and 80μmol / L lysine) and control group without lysine, respectively. The supernatants were harvested 24 and 48 hours respectively, The proliferation of cells was detected by MTS method. The expression of HBsAg and HBeAg was detected by ELISA. The copy number of HBV DNA and the expression of miR-210, miR-185, miR-199a-3p, miR-370, miR-196a, miR-184, miR-217. The HepG2.2.15 cells were transfected with miR-210, miR-185, miR-199a-3p, miR-370, miR-196a, miR-184 and miR-217 antisense strands respectively Activity, ELISA detection of HBsAg and HBeAg expression levels. Results In the absence of an effect on the proliferative activity of HepG2.2.15 cells, lysine inhibited the production of HBsAg and the proliferation of HBV. The expression of miR-210, miR-185 and miR-199a-3p was significantly inhibited by 20μmol / L L-Rhein, while the expression of miR-370 was inhibited. Compared with the control group, the difference was statistically significant (P < 0.05). Conclusion L-Rhein may inhibit the proliferation of HBV and the production of HBsAg in HepG2.2.15 cells by affecting the expression of miRNA in cells.