乙型肝炎病毒不同基因区RNA干扰抑制乙型肝炎病毒复制和表达的实验研究

目的针对乙型肝炎病毒(HBV)不同基因区设计siRNA,观察其对HBV表面抗原、e抗原分泌和HBVDNA复制的影响。方法靶向HBV基因区S、C、X的siRNA分别转染可稳定分泌HBV颗粒的HepG2.2.15细胞,转染后72h,采用ELISA方法检测上清液中HBsAg、HBeAg的含量,Real-timePCR定量检测HBVDNA拷贝数。结果靶向不同基因区的siRNA均能不同程度地抑制HepG2.2.15细胞HBsAg、HBeAg的分泌和HBVDNA的复制,其中S区siRNA对HBsAg呈现非常显著的抑制作用,抑制率为91.88%(P﹤0.01),对HBeAg表达的抑制作用较弱,抑制率为24.85%(P﹤0.05),对HBVDNA的抑制率为63.07%(P﹤0.01);C区siRNA对HBeAg和HBVDNA抑制作用较强,分别为54.77%(P﹤0.01)和76.97%(P﹤0.01),但对HBsAg的表达无明显影响(P﹥0.05);X区siRNA对HBsAg和HBeAg、HBVDNA均有抑制作用,抑制率分别为90.83%(P﹤0.01)、34.85%(P﹤0.01)和70.31%(P﹤0.01)。靶向不同基因区的siRNA对HBsAg、HBeAg、HBVDNA的抑制作用在一定的程度上存在剂量效应,而无关序列对HBsAg、HBeAg和HBVDNA的复制和表达几乎无干扰作用(P﹥0.05)。结论靶向HBVC、S、X基因区的siRNA可特异、高效、稳定地抑制HBV的复制和表达,为应用RNA干扰治疗乙型肝炎病毒奠定了一定基础。

siRNAs targeting different gene regions of hepatitis B viral efficiently inhibit HBV replication and HBsAg and HBeAg expression in vitro

OBJECTIVE To design small short interfering RNAs(siRNA)targeting hepatitis B viral different gene regions,and evaluate inhibitory effect of siRNAs on hepatitis B viral replication and HBsAg and HBeAg expression in vitro. METHODS siRNAs targeting S,C,and X regions on HBV genome were transfected into HepG2.2.15 cells,a human hepatoblastoma cell that constitutively produces HBV particles,and after 72 h post-transfection,the culture medium was collected. Enzyme-linked immunosorbent assay was used to measure the HBsAg and HBeAg levels in culture medium and HBV DNA replication was determined by real-time fluorescence quantitative PCR. RESULTS siRNAs targeting hepatitis B viral different gene regions could all inhibit hepatitis B viral replication and HBsAg and HBeAg expression at different extent. siRNAs targeting S region could markedly suppress the expression of HBsAg with 91.88%(P﹤0.01),but less inhibitory effect on the HBV DNA replication and the expression of HBeAg,with inhibitory rates of 63.07%(P﹤0.01)and 24.85%(P﹤0.05),respectively;siRNAs targeting C region could efficiently suppress the expression of HBeAg and HBV DNA replication with 54.77%(P﹤0.01)and 76.97%(P﹤0.01),but no effect on the expression of HBsAg(P﹥0.05);siRNAs targeting X region could all inhibit HBsAg and HBeAg expression and HBV DNA replication,with inhibitory rates of 90.83%(P﹤0.01),34.85%(P﹤0.01)and 70.31%(P﹤0.01),respectively. siRNA targeting HBV different gene regions exerted robust inhibition on HBV replication and antigen expression in a dose-dependent manner at different extent,whereas the irrelevant siRNA control did not show any inhibitory effect on the replication and expression of HBV. CONCLUSION These results demonstrated that siRNA targeting HBV different gene regions can consistently inhibit the replication and expression of HBV with great potency and specificity in vitro,which suggestsed that RNAi strategy may represent a potentially efficacious approach to the clinical management of HBV infection.