Construction of NTCP-Huh7 cell lines and their susceptibility to HBV / 医学研究生学报

ABSTRACT

ObjectiveThe transient expression of the functional receptor sodium taurocholate cotransporting polypeptide (NTCP) leads to the inefficiency and instability of the hepatitis B virus (HBV)-associated hepatocellular carcinoma cell model. The aim of this study was to construct NTCP-Huh7 cell lines by transfecting GV358-NTCP into Huh7 cells and identify their susceptibility to HBV.MethodsThe recombinant plasmid GV358-NTCP was obtained by ligation of GV358 and NTCP, and then transfected into Huh7 cells for the construction of NTCP-Huh7 cells. The NTCP-Huh7 cells (NTCP-Huh7 group) and Huh7 cells (Huh7 group) were infected with HBV and co-incubated with HBV for 18 hours, and the co-incubation was continued after change of the culture medium. At 2, 4, 6, 8, 10, and 12 days of incubation, the supernatant and cells were collected for measurement of the contents of HBsAg, HBeAg, HBcAg and HBV DNA in the supernatant and HBV cccDNA in the cells as well as for determination of HBV susceptibility of the NTCP-Huh7 recombinant cells.ResultsWestern blot showed stably expressed NTCP proteins in the NTCP-Huh7 cells. At 8 days of incubation, the levels of HBsAg and HBeAg were significantly higher in the NTCP-Huh7 group (0.866±0.040 and 0.603±0.053) than in the Huh7 group (0.237±0.063 and 0.209±0.112) (P<0.05), reaching the peak at 8 days and also remarkably higher in the former than in the latter group at 4, 6 and 10 days (P < 0.05). So were the levels of HBV DNA and HBV cccDNA in the NTCP-Huh7 than in the Huh7 group at 4, 6, 8, 10 and 12 days (P < 0.05). Immunofluorescence assay revealed the core antigen of HBV in the NTCP-Huh7 but not in Huh7 cells.ConclusionNTCP-Huh7 cells obtained by transfection of the GV358-NTCP recombinant plasmid into Huh7 cells are susceptible to HBV infection.