Establishing The Hybrid Cell Line For HBV

BackgroundThe hepatitis B virus(HBV), is one of the major pathogens which can cause severe liver disease. It was reported by WHO, globally, more than 2 billion people were infected by HBV. And there are about 350 million chronic carriers of HBV. The chronic hepatitis B can not be cured, What's more, hepatitis B can deteriorate to cirrhosis and hepatocellular carcinoma with the development of illness. From Blumberg having found HBV surface antigen in 1965 to having completed clone and sequence of HBV DNA genome in 1997, researchers have been making great progresses in constitution of HBV genome, structure and biological traits of antigens, and life circle of HBV. Due to deficiency of exoteric multiplying system and pragmatic cell models, little was known about the early mechanisms of initiation in infection. Consequently, basic and clinical research on hepatitis B and other types of hepatitis concerned were hampered some way. Owing to the finiteness of human hepatic tissue, the restriction of HBV adhesion, DNA transcription and replication host, at the same time prior cell models are all have their inevitable defects, we need to establish a better cell model. This model should be both suitable for transfection and sensitive to virus particle from blood serum. The cell should be similar to human hepatic cell in morphology and can express HBV antigens stably.ObjectiveTo establish a hybrid cell that is different from either HepG2 cell or human primary hepatic cell. This cell carries HBV, and can be serial subcultivation in vitro.MethodsHGPRT defect HepG2 cell, which was induced by 6-MP, was fused with human primary hepatic cell infected by hepatitis B virus. After being screened with HAT, the hybrid cell was cloned through limiting dilution assay. Subsequently, the hybrid cell was identified by karyotype analysis. HBV DNA and HBsAg,HBeAg were examined at different generations. HepG2 and human primary hepatic cell infected by hepatitis B virus were taken as control groups.ResultsThe hybrid cell is similar to HGPRT defect HepG2 cell morphologically and can be subcultured in vitro. The Karyotype analysis results show that the modal chromosome numbers are 99 in the hybrid cells, indicating that the hybrid cells contain all genomic factors from both HepG2 and human hepatic cell. HBV DNA can be detected in the cells and supernates by nest PCR. In the supernates HBsAg and HBeAg can be detected too. Conceivably the hybrid cell possesses the replication a generation in vitro of HepG2 as well as the sensitivity of human primary hepatic cell to HBV, which paves secretion of HBV DNA.ConclusionAs a new hybrid cell line, it has the characteristics of both generation in vitro of HepG2 and sensitivity of human primary hepatic cell to HBV, which paves way for further study of HBV infection cell model.