乙型丙型肝炎病毒合并感染细胞模型的建立

Establishment of a HBV and HCV co-infection cell model

目的:利用慢病毒载体在可感染丙型肝炎病毒(hepatitis C virus,HCV)的Huh7.5.1细胞中过表达人肝细胞乙型肝炎病毒(hepatitis B virus,HBV)受体钠离子牛磺胆酸共转运蛋白(sodium taurocholate cotransporting polypeptide,NTCP)编码基因,建立Huh7.5.1-hNTCP细胞模型,使其可以被HBV、HCV共同感染。方法:使用携带人NTCP编码基因、绿色荧光蛋白(green fluorescent protein,GFP)基因、嘌呤霉素抗性基因的GV358重组慢病毒载体,以感染复数(MOI)50感染Huh7.5.1细胞。经过嘌呤霉素筛选获得稳定表达人NTCP基因的Huh7.5.1-hNTCP细胞株,并通过Western blot验证NTCP蛋白的表达。使用HepAD38细胞来源的HBV感染Huh7.5.1-hNTCP细胞,通过ELISA及qPCR等方法检测HBV感染后不同时间点乙肝病毒表面抗原(HBsAg)、e抗原(HBeAg)的表达以及HBV DNA的复制。HBV感染后24 h予以HCV-JFH1感染,检测合并感染状态下HBV DNA以及HCV RNA的复制。结果:Western blot结果表明Huh7.5.1-hNTCP细胞株可稳定表达HBV受体NTCP。HBV感染Huh7.5.1-hNTCP细胞后ELISA、qPCR结果显示,HBsAg、HBeAg、HBV DNA合成逐渐增加,并在第9天达峰。HBV/HCV合并感染Huh7.5.1-hNTCP细胞后,qPCR检测结果显示不同时间点HBV DNA、HCV RNA复制逐渐增加。结论:建立了Huh7.5.1-hNTCP细胞模型,该模型可被HBV/HCV合并感染。

Objective:To establish a sodium taurocholate cotransporting polypeptide(NTCP)-complemented Huh7.5.1 cell line(Huh7.5.1-hNTCP)that supports hepatitis B virus(HBV)and hepatitis C virus(HCV)co-infection. Methods:Huh7.5.1 cells were infected with GV358 recombinant lentiviral vector carrying human NTCP encoding gene,green fluorescent protein(GFP)gene and puromycin resistance gene with the multiplicity of infection(MOI)of 50. NTCP positive cells were obtained through the puromycin screening and NTCP expression was identified by Western blotting. Huh7.5.1-h NTCP cells were infected with HBV derived from HepAD38 cells. The expression of HBsAg,HBeAg and HBV DNA at different time points were detected by ELISA and qPCR. HCVJFH1 was added 24 hours after HBV infection,and then HBV DNA and HCV RNA were detected in co-infected cells. Results:Western blotting results indicated that Huh7.5.1-hNTCP cell line stably expressed the HBV receptor NTCP. ELASA and q PCR results showed that HBsAg,HBeAg and HBV DNA secretion increased gradually and peaked on the 9 thday in HBV infected Huh7.5.1-hNTCP cells. The results of qPCR suggested that the replication of HBV DNA and HCV RNA increased gradually at different time points in HBV and HCV co-infected cells. Conclusion:Huh7.5.1-hNTCP cell line was established,and it can be co-infected by HBV and HCV.