pGenesil-siHBV X 对 HepG2.2.15 细胞 HBV 表达和复制的抑制效果研究

 目的 研究针对HBV X基因区设计的小干扰RNA（siRNA）表达载体质粒pGenesil-siHBV X 对HepG2.2.15细胞HBV表达和复制的抑制效果及特异性。方法 针对HBV X区设计siRNA表达载体质粒pGenesil- siHBV X。分别用培养液（空白对照）、脂质体Metafectene、pGenesil 空载体、pGenesil-siHK（阴性对照）、pGenesil-siAFP（特异性对照）、pGenesil-siHBV X处理或转染HepG2.2.15细胞各3次。于每次转染后24 h，取各组细胞培养上清液，用时间分辨荧光免疫测定技术检测上清液中HBsAg和HBeAg含量，用化学发光法检测AFP含量，用PCR荧光定量技术检测HBV-DNA复制水平。结果 pGenesil-siHBV X转染能抑制HepG2.2.15细胞对HBV标志物的表达，且抑制作用随转染次数增加而增强。第3次转染后，pGenesil-siHBV X组细胞上清液中 HBsAg、 HBeAg和HBV-DNA检测结果分别为（6.26 ± 1.07）ng/ml 、（0.13 ± 0.05）Ncu/ml和（3.01 ± 0.40）×107拷贝/ml，与空白对照组的（22.50 ± 1.39）ng/ml、（1.12 ± 0.11）Ncu/ml和（12.33 ± 1.28）×107拷贝/ml比较，差异有统计学意义（t值分别为12.80、12.21、9.71，P < 0.05）；pGenesil-siHBV X 转染不影响细胞对AFP的表达（t = 0.18，P = 0.86）。结论 pGenesil-siHBV X可以有效和特异地抑制HepG 2.2. 15细胞HBV-DNA的复制及HBsAg和HBeAg表达。

Suppressing effect of pGenesil-siHBV X on the expression and replication of hepatitis B virus in HepG2.2.15 cells

Objective To evaluate the effect of pGenesil-siHBV X on the replication and expression of hepatitis B virus in HepG2.2.15 cells. Methods HepG2.2.15 cells were cultured in six-well plate and treated 1 to 3 times with the culture fluid (blank control group), METAFECTENE, pGenesil, PGenesil-HK (negative control group), PGenesil-siAFP (specific control group), and pGenesil-siHBV X. After been treated for 24 hours, the expression of HBV antigen in the supernatant of all the groups were determined with Time-resolved Immunofluorometric Assay kit (TRFIA), the level of AFP was detected by chemiluminescent method, and the level of HBV-DNA was examined by fluorescence quantitative PCR. Results pGenesil-siHBV X could effectively and specifically inhibit the levels of HBsAg, HBeAg, and HBV-DNA, and the inhibiting effects increased with the times of transfection. Compared to the blank control group, the levels of HBsAg, HBeAg, and HBV-DNA in the supernatant in the pGenesil-siHBV X group decreased obviously after 3 times of transfection [(22.50 ± 1.39) ng/ml, (1.12 ± 0.11) Ncu/ml, and (12.33 ± 1.28) × 107 copies/ml vs. (6.26 ± 1.07) ng/ml, (0.13 ± 0.05) Ncu/ml, and (3.01 ± 0.40) × 107 copies/ml; t = 12.80, 12.21 and 9.71; P < 0.05]. Furthermore, pGenesil-siHBV X did not suppress the expression of AFP gene (t = 0.18, P = 0.86). Conclusion pGenesil-siHBV X could effectively and specifically inhibit HBV replication and expression of HBsAg and HBeAg in HepG2.2.15 cells.