Study On HBV Infection For Primary Hepatocytes In Macaca Fascicularis

HBV infection,especially chronic HBV infection,can lead to hepatitis,liver cirrhosis and even hepatic carcinoma.HBV has become one of the most serious diseases threaten to public health.About HBV infection,kinds of models in vitro and in vivo have been constructed.Although these models are used to explore the mechanism of HBV infection and develop new anti-HBV drugs succesfully,there is still lack of HBV infection models similar to human.Since NTCP was confirmed as an essential functional receptor in HBV infection,NTCP has become a new target for anti-HBV drugs and a popular topic to help to construct novel HBV infection models.In this study,we attempted to explore whether human NTCP could promote HBV infection in primary macaca fascicularis hepatocytes.First,human NTCP was transferred into HepG2 and Huh7 cells by lentivirus and the expression of human NTCP was verified by TaqMan Probe real-time PCR of DNA,cDNA(RNA)and western blotting.Then HepG2 and Huh7 cells expressing human NTCP were co-cultured with HBV serum,cell culture medium was collected and the HBsAg in the supernatant was quantitated by enzyme linked immunosorbent assay kit.The results showed that HBsAg elevated significantly and suggested that HBV was susceptible to HepG2 and Huh7 cells expressing human NTCP.Second,human NTCP was transferred into macaca fascicularis hepatocytes with the same method as HepG2 cells.The results showed that human NTCP couldn’t be expressed in primary hepatocytes,probably due to suppression by the endogenic macaca fascicularis NTCP.At last,human NTCP was transferred into hepatocytes followed inactivation of the macaca fascicularis NTCP which was knockout by CRISPR/Cas9 system.Based on the real-time PCR results of DNA and cDNA(RNA),we could deduce that human NTCP could be expressed in primary hepatocytes.After that,HBV sera were added into the cell culture medium and HBsAg was quantitated as same to HepG2 cells.However,the ELISA results of HBsAg showed that HBV life cycle was restricted in macaca fascicularis hepatocytes expressing human NTCP.In a word,our data showed that expression of human NTCP promoted HBV infection in HepG2 and Huh7 cells,but doesn’t work in macaca fascicularis hepatocytes,and it indicated that human NTCP was insufficient to introduce HBV infection in primary macaca fascicularis hepatocytes,as well as co-effectors was essential and needed further exploring.