体外针对X基因的小干扰RNA的抗乙型肝炎病毒效应研究

Inhibitory Effect of Small Interfering RNA Targeting X Gene on Hepatitis B Virus Replication

目的:探讨体外针对乙型肝炎病毒(HBV)X基因的小干扰RNA(siRNA)对HBV复制和抗原表达的抵制作用。方法:利用siRNA表达框架法设计针对HBV X基因的siRNA,转染HepG2.2.15细胞,RT-PCR半定量检测转染前后X基因的表达;ELISA法测定各组24、48、72h HBsAg和HBeAg的含量;荧光定量PCR检测48h时HBV DNA的变化。结果:制备了HBV X基因的siRNA,转染后24、48和72h,HBV X基因mRNA的量分别减少了57%、78%和40%;siRNA能抑制HBsAg和HbeAg的分泌,抑制高峰在48h,抑制率分别为42%和43%;荧光定量PCR证实HBV DNA的复制亦受到抑制。结论:针对HBV X基因的siRNA在体外具有抑制HBV复制和抗原表达的作用。

Objective： To explore the inhibitory effect of small interfering RNA（siRNA） targeting the X gene sequence of hepatitis B virus（HBV） in vitro on HBV replication and its antigen expression. Methods： siRNA targeting HBV X gene （HBVx） mRNA were designed and generated by PCR amplification and siRNA expression cassettes （SEC） methods. The product of PCR was transfected into HepG2.2.15 cells using LipofectAMINE 2000 reagent. The expression levels of HBVx mRNA at different transfection times were assayed by RT-PCR. At 24, 48 and 72 hours after transfection, the levels of HBsAg and HBeAg in the cell culture supernatant were determined with ELISA kits. The HBV DNA in the supernatant was also determined by fluorogenic quantitative PCR（FQ-PCR）. Results： The method of using SEC to generate siRNA was successfully. HBVx mRNA expression in the cells treated by HBVx-siRNA at 24, 48 and 72 h were significantly reduced by about 57%, 78% and 40% respectively. HBVx-siRNA had shown to efficiently and specifically inhibit the synthesis of surface antigen and e antigen of I-IBV, with inhibitory rate of 43% and 42%, peaking at 48 h after transfection. FQ-PCR also showed the replication of HBV DNA was reduced to lower levels. Conclusion： Results in this experiment demonstrates that the short interfering RNA targeting HBVx gene exert s robust inhibition on HBV replication and antigen expression.