乙型肝炎病毒X基因siRNA对HepG2．2．15细胞的影响

Effect of si RNA on hepatitis B virus X gene to HepG2.2.15 cells

目的:利用化学合成的小干扰RNA(si RNA)转染HepG2.2.15细胞,构建针对乙型肝炎病毒(HBV)X基因的细胞干扰模型,研究其在体外对HBV复制和抗原表达的抑制作用。方法 :将构建成功的HepG2.2.15细胞干扰模型,于转染后24、48、72 h,化学发光免疫法检测细胞上清液中HBsAg、HBeAg,ELISA法检测细胞上清液HBxAg蛋白表达量,荧光定量PCR检测转染后细胞中HBx mRNA相对表达量,CCK-8法检测细胞增殖能力。结果:HBx-siRNA转染HepG2.2.15细胞后,细胞增殖能力被抑制,细胞中HBx mRNA及上清液中HBxAg的表达量下降(P<0.05);并发现其能抑制细胞HBsAg、HBeAg表达,抑制高峰在72 h,抑制率分别是66%和58%;荧光定量PCR证实细胞上清液中HBV DNA的表达量下降。结论:成功地构建了HBV X基因的Hep G2.2.15细胞干扰模型,在体外具有抑制Hep G2.2.15细胞增殖和HBV基因的复制与表达的作用。

Objective Using chemically synthesized small interfering RNA （siRNA） transfected HepG2.2.15 cells to construct a cell model in interfering hepatitis B virus （HBV） X gene, studying the inhibi- tion of HBV replication and antigen expression in vitro. Methods After transfection of HepG2.2.15 cell for 24 h, 48 h, 72 h, detecting the cell supernatant of HBsAg and HBeAg by chemiluminescence immunoassay, the cell supernatant HBxAg protein by ELISA, the HBx mRNA relative expression of transfected cell was detected by fluorescence quantitative polymerase chain reaction （PCR）, the ability of cell proliferation was detected by CCK- 8 assay. Results After HBx-siRNA transfeeted HepG2.2.15 cells, cell proliferation ability was inhibited. The cell of HBx mRNA and the cell supernatant of HBxAg expression decreased （P 〈 0.05）; at the same time it in- hibited the expression of HBsAg and HBeAg. The suppressed peak and the inhibition rate were 66% and 58% respectively at 72 h. The fluorescence quantitative PCR confirmed that expression of HBV DNA in the super- natant was decreased. Conclusion The HepG2.2.15 cell interference model ofHBv x gene has been success- fully constructed, which has an effect of inhibiting proliferation of HepG2.2.15 cells and replication and expres- sion of HBV gene in vitro.