多种小分子干扰RNA联合抑制乙型肝炎病毒的体外研究

小分子干扰RNA(siRNA)能够在哺乳动物细胞中引起包括病毒基因在内的基因沉默。为了研究多种siRNA联合应用在体外抑制乙型肝炎病毒(HBV)复制中的效果,本研究设计了12种针对不同HBV靶点的siRNA,转染可稳定分泌HBV颗粒的HepG22.2.15细胞,并采用了酶联免疫法检测上清液中HBsAg和HBeAg的含量,实时定量PCR法检测细胞中HBV的DNA含量。结果发现这12种分子均能在不同程度上抑制病毒复制。进一步研究表明它们对HBV的抑制作用在一定程度上存在剂量效应和协同作用,单分子siRNA在80nmol/L处对HBsAg和HBeAg的抑制率分别可达到约80%和60%,而多分子siRNA组合在20nmol/L处对HBsAg就能达到90%的抑制率,但对HBeAg表达的抑制率提高不明显。单分子siRNA在80nmol/L处对HBVDNA复制的抑制率可达到90%以上,而多分子siRNA组合在20nmol/L处对DNA含量就能达到约90%的抑制率。本研究的结果为进一步开发新的联合应用多种siRNA治疗HBV的途径打下了基础。

INHIBITION OF HEPATITIS B VIRUS BY COMBINED siRNAs IN VITRO

Small interfering RNA (siRNA) technology is a powerful tool to knockdown gene expression in mammalian cells including genes of viral origin. To study the inhibition of hepatitis B virus (HBV) by combined siRNAs in vitro, HepG2 2.2.15, a human hepatoblastoma cell that constitutively produces HBV particles, was transfected with 12 siRNAs that were designed to tar- get different regions on HBV genome. Enzyme-linked immunosorbent assay was used to measure the HBsAg and HBeAg levels in culture media, and HBV replication was determined by real-time quantitative PCR. The results showed that HBV replication could be inhibited by treatment with these siRNAs in a dose-dependent manner. The expression of HBsAg was inhibited by 80% and 90% with 80nmol/L single siRNA treatment and 20nmol/L combined siRNAs treatment, respec- tively. HBeAg was inhibited by about 60% with single siRNA treatment, and the inhibition didn't increase much in the case of combined siRNAs treatment. HBV replication was inhibited by 90% with 80nmol/L single siRNA treatment or 20nmol/L combined siRNAs treatment. These data pro- vide solid evidence for developing new approaches in HBV treatment with combined siRNAs.