联合应用siRNA和拉米夫定抑制HBV复制和表达的实验研究

摘要

The inhibition of the combined application of small interferent RNA (siRNA) and lamivudine against HBV antigen expression and replication in HepG 2. 2. 15 cell was observed. Establish and transfect recorabinant plasmid psil-HBV into HepG 2.2. 15 cell. The cell culture medium of the transfected cell was added with lamivudine (0.05 μm) , and respectively harvested the cells at 48 h, 72 h, and 96 h. Examined HBeAg and HbsAg with ELISA; the level of HBV DNA was tested with real time PCR; the level of HBV mRNA was tested with inverse transcription PCR. The results showed that 96 hours after the combined application of siRNA and lamivudine the inhibtion rate of HbeAg and HbsAg in the cell culture broth were 91.8% and82.4% respectively (P<0.05), the expression level of HBV mRNA noticeably reduced. Therefore, it is more effective to apply siRNA in combination with lamivudine than to apply siRNA singly in HepG 2. 2. 15 cell to inhibit HBV replication.%观察联合应用小干扰RNA和拉米夫定对HepG2.2.15细胞中HBV抗原表达和复制的抑制作用.构建并转染重组质粒psil-HBV到HepG2.2.15细胞中.转染后的细胞培养基中加入拉米夫定(0.05 μm),分别于48、72、96 h收获细胞.用ELISA方法检测HBeAg和HBsAg；HBV DNA水平用实时定量PCR测定；用逆转录PCR检测HBV mRNA水平.96 h后联合应用小干扰RNA和拉米夫定组细胞培养上清中HBeAg和HBsAg抑制率分别为91.8％和82.4％ (P ＜0.05)；HBV mRNA表达水平明显降低.HepG2.2.15细胞中联合应用小干扰RNA和拉米夫定对HBV复制的抑制作用比单独应用siRNA或拉米夫定更有效.