以S区为靶位的小干扰RNA抗乙型肝炎病毒的实验研究

目的以乙型肝炎病毒（HBV）S基因区为靶位,构建表达siRNA的质粒载体pSilencer3．1-H1hygro,体外观察siRNA抗HBV的效果。方法以HepG2．2．15细胞为靶细胞,利用脂质体Metafectene转染表达siRNA的质粒载体pSilencer3．1-H1hygro于细胞中,用时间分辨免疫荧光分析法（IFMA法）检测细胞上清中HBsAg和HBeAg,用定量聚合酶链反应（FQ—PCR）检测细胞上清DNA,用逆转录（RT）-PCR检测HBV mRNA。结果成功构建了表达siRNA的转录质粒载体,siRNA可抑制HBV的抗原表达和病毒复制,1、2、4μgsiRNA对HBsAg的抑制率分别为75％、82％、89％;对HBeAg的抑制率分别为32％、38％、43％;对HBVDNA的抑制率分别为30％、43％、49％;对HBVRNA的抑制率分别为30％、70％、90％。结论靶向HBVS区的siRNA能抑制HBV的抗原表达和复制;siRNA抑制作用呈剂量依赖性和序列特异性。

Inhibition of hepatitis B virus replication and expression by RNA interference in vitro

OBJECTIVE: To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that target HBV S gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro. METHODS: HepG2.2.15 was used as target cell. The plasmid expressing small interfering RNA was transfected into the cultured cells via liposome metafectene, HBsAg and HBeAg were analyzed by time-resolved immunofluorometric assay, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV S-mRNA was detected by semi-quantitative RT-PCR. RESULTS: The plasmid expressing siRNA was successfully constructed. The S region siRNAs could effectively inhibit both antigens secretion and HBV replication compared with controls, HBsAg levels decreased by 75%, 82%, 89%; HBeAg levels decreased by 32%, 38%, 43%; HBV DNA production decreased by 30%, 43%, 49%; The HBV mRNA species was reduced by 30%, 70%, 90% when transfected with 1 microg, 2 microg, 4 microg HBV S-siRNA, respectively. CONCLUSION: These results demonstrate that RNAi can substantially inhibit HBV replication and the antigens expression in the infected cells. These inhibitive effect of siRNA on HBV was dose-dependent and sequence-specific.