Establishment Of A Novel Cell Model Of Hepatitis B Virus Infection

BackgroundThe infection of Hepatitis B virus is worldwide significant problems which can affect human health,China is highly endemic areas,where the HBsAg positive rate of the general population is 9.09%, and there are about 120 million chronic carriers of HBV,nearly 1/3 proportion of all chronic carriers in the world. At present,though people own a considerable in-depth understanding of HBV and the corresponded diseases, the biological research and treatment of HBV is progressed slowly because of the lack of suitable animal model and in vitro cell culture system. Currently there are two cell models used for the study of HBV in vitro,primary human hepatocytes and hepatoma-derived cells.The primary human hepatocytes retain important cellular characteristics of differentiated hepatocytes,but they begin to dedifferentiate and lose the capability to support HBV infection and replication within several days,which limit the application of them.Hepatoma-derived cells which are transfected HBV genomes or added chemical reagents rather than the way of natural infection of HBV into the cell,so can not be used for the early infection of HBV. Thereby, we have attempted to establish an in vitro infection cell culture system, which can infect HBV and be serial sub-culture in vitro.It could be a useful tool for investigating the mechanisms of the early stages in HBV infection and analyzing the in vivo process of viral infection and viral life cycle in human hepatocytes.ObjectiveTo establish a hybrid cell line by fused HepG2 cells with primary human hepatocytes,which can infect the hepatitis B virus(HBV) and be serial sub-culture in vitro.Evaluate the ability of the hybrid cell line to HBV natural infection.MethodsIsolation and culture of human primary hepatocytes without carrying HBV,then,the human primary hepatocytes were fused with HGPRT-HepG2 which were induced by EMS.The hybrid cells were identified by the trypsin G-banding method.Co-incubated the hybrid cells and HepG2 cells with serum-derived HBV virions respectively.Detected the intracellular and secreted HBV DNA as well as the intracellular HBV cccDNA by nested PCR.HBcAg in infected cells were analyzed by indirect immunofluorescence.HBsAg and HBeAg in the supernatants of infected cells were identified by electrochemiluminescence.ResultsA hybrid cell line was established successfully by fused HepG2 cells with primary human hepatocytes,which can be sub-cultured in vitro. The Karyotype analysis results showed that the modal chromosome numbers of the hybrid cells were 99,HBV DNA was detected both in the hybrid cells cells and in the culture media consistently after days 4 post infection.HBV cccDNA was detected consistently after days 3 post infection.HBcAg,HBsAg and HBeAg were detected consistently after days 4 post infection.However,the results of the infected HepG2 were negative.ConclusionA new hybrid cell line was established successfully.The new hybrid cell line inherited the characteristics of both HepG2 and human primary hepatocyte,and can be used for establishing an in vitro HBV infection cell model.