KRT8对HBV复制影响的体外研究

An in Vitro Study of the Effect of KRT8 on the Replication of Hepatitis B Virus in HepG2.2.15 Cells

目的:研究宿主蛋白KRT8对HBV复制的影响.方法:体外培养稳定表达HBV的肝癌细胞株(HepG2.2.15),分别用干扰KRT8基因的siRNA转染HepG2.2.15细胞和高表达KRT8基因的质粒转染HepG2.2.15细胞.RT-PCR验证转染效率,荧光定量PCR检测转染48小时后的细胞上清液HBV DNA水平.用拉米夫定抑制HepG2.2.15细胞HBV复制,RT-PCR检测基因表达水平.对数据采用t检验和方差分析,p<0.05为差异有统计学意义.结果:干扰KRT8基因表达后,HepG2.2.15细胞上清液中HBV DNA水平与对照组相比下降34%～38%(p<0.05).高表达KRT8基因后,HepG2.2.15细胞上清液中HBV DNA水平是对照组的28倍(p<0.01).抑制细胞HBV DNA复制后,HepG2.2.15细胞KRT8mRNA水平与对照组相比,下降了24%,但统计学无明显差异.结论:抑制宿主KRT8基因表达具有抗病毒作用.

Objective. To investigate the effect of host protein KRT8 on HBV DNA replication. Methods. Hepatitis B virus （HBV）-transfected human hepatoblastoma cell line HepG2.2.15 was cultured in vitro. We used siRNA to silence KRT8 gene expression and plasmid vector to up regulate KRT8 gene expression and transfected them to HepG2.2.15 cells respectively. Forty-eight hours after the transfection, the cells were harvested for detecting the level of KRT8 gene expression and the culture supernatants were collected for measuring the level of HBV DNA replication. Lamivudine was selected to inhibit HBV DNA replica- tion and RT-PCR was chosen to detect the level of KRT8 gene expression. Statistical analysis was per- formed using the t test or ANOVA, when appropriate. A result of P〈0.05 was considered statistically significant. Results. When KRT8 mRNA was suppressed, the HBV DNA level in RNA interference groups was decreased by 34%-38% compared with that of the control group （P〈0.05）. When KRT8 mRNA was over-expressed, the HBV DNA level was increased in vector group, 28 times that of the con- trol group （P〈0.01）. When HBV DNA replication was inhibited, no significant difference was found be- tween the lamivudine group and the control group in their HBV DNA level （a decline of 24%）. Conclu- sion. HBV DNA replication could be inhibited by suppressing KRT8 mRNA expression.