Infection Of Transgnic Adult Hepatocytes Expressing Human Tert With Hepatotropic Viruses

The hepatitis B virus (HBV) and hepatitis C virus (HCV), which cause acute or chronic viral hepatitis and other liver diseases including cirrhosis and hepatocellular carcinoma (HCC), are hepatotropic viruses that represent a serious global health problem. The number of chronically infected subjects is estimated at 360 million for HBV and 170 million for HCV and annually these pathogens kill more than 1.5 million people worldwide.In the past decade substantial progress has been made in treating these chronic infections as well as bring many problems.The side effects of treatment and emergence of variants defying therapeutic treatment are major problems.Therefore developing new antiviral compounds,which are more efficacious,little side effect and better tolerated, are emergent.The key is to find cell culture systems besides animal models for antivirus compounds selected and vaccines evaluated.Infection normal human hepatocytes or fetal hepatocytes with serum-derived HBV or HCV are the ideal system in which to study hepatotropic virus infectivity. Differentiated phase affected the susceptibility of cells to virus infection. Accordingly, it was found that only human hepatocyte primary cultures were susceptible to both HBV and HCV infection,which facilitated systematic investigations of virus early event and consequently innate immune response.Human hepatocytes belong to well-differentiated epithelioid cells. When cultured in vitro, however, they proliferate poorly and divide only a few times, eventually result in senescence or death. Continuous proliferation even immortalized could be achieved however by introducing oncogenes. Telomeres are nucleoprotein complexes at the chromosome ends which play a critical role in the maintenance of chromosomal integrity.The telomere lengths is relevant to cell survival.Progressive telomere shortening with each cell division eventually triggers an alteration in telomere structure, which cause senescence or apoptosis from the perspective. Telomerase is a specialized RNA-protein complex that which responsible for the de novo synthesis and maintenance of telomere repeats.Telomerase expression is low or absent in most human somatic tissues, except for tissue stem cells, germ cells, immortalized cells and tumor cells. The telomerase reverse transcriptase(TERT) is core subunit, which regulate telomerase activity strictly.The human somatic cells transducted with gene encoding human TERT(hTERT) can escape p53-mediated senescence or other checkpoint responses,thus prolong in vitro culture time.In order to answer this question, the aim of the study was established. In view of limited availability and difficult culture, human hepatocyte clones were developed by transduction with the gene encoding the hTERT. The transgenic cells infectivity was assayed by exposing HBV-positive serum to both cell and animal level. The transgenic cells were useful tool for selecting antivirus compounds and evaluating vaccines against HBV, which lay the foundation for further study on developing models of HCV infection too.The main contents and results of the study are discussed below.1. Isolation and culturing of human hepatocytes from rejected livers. The human hepatocytes were isolated from liver fragment using modified"two-step"collagenase procedure.The cells were assessed for morphological characters as well as metabolism traits when culture in vitro. After seven days in culture, the human hepatocyte clones were appeared. The cells morphology was same as adult human hepatocytes. The hepatocyte-specific markers were analyzed by RT-PCR.The isolated primary hepatocytes displayed expression of ALB, AAT, HGF, CK18, TF, GS except for AFP.The isolated primary hepatocytes were the same as the freshly isolated primary hepatocytes. Trace albumin detection showed the albumin secretion increased from day 1 to day 9,which reach a peak on day 9 followed by descent corresponding to cell growth.2. Screening and identification of positive transfected cells. The recombinant plasmids were transfected into packaging PT67 cells using Lipofectamine 2000 according to provided protocol. Virus-producing cells were selected after two weeks G418-selection. The cultured human primary hepatocytes were infected with viral supernatants harvested. The transgenic human primary hepatocytes expressing hTERT were established, named HC-T. The transgenic human primary hepatocytes displayed expression of ALB, TF, GS except for AFP. Immunofluorescence staining showed CK18 was located in the cytoplasm.Glycogen storage detection indicated that plentiful glycogenic granules deposited in the cytoplasm.Morphologic, phenotypic and function analysis proved that the transgenic human primary hepatocytes had the same features as the freshly isolated primary hepatocytes.3. HBV infection of the transgenic human primary hepatocytes. As an infectious inoculum, the serum-derived HBV was selected to infect HC-T. HC-T cells were incubated with HBV positive serum. The HBsAg and HBV DNA in culture supernatant, passage supernatant and reinfection supernatant was analyzed by ELISA and Real-time PCR individually. HC-T cells did share many characteristics with normal human hepatocytes including morphologic, phenotypic, function aspects and HBV infectivity. HC-T infectivity to HBV was showed by the kinetics of HBsAg and HBV DNA .After entering into HC-T, HBV began to replicate. After a complete replicative cycle, the virus partical was released into supernatant at 2 days post inoculation ( pi). HBV was detected with cyclic fluctuations.The low level of HBV protein and DNA were relative to HBV persistent. The HBV certain protein may inhibit cells apoptosis, which need further study.The virion released into supernatant had infectivity and infective patter was similar to serum-derived HBV with cyclic fluctuations. Repetitive analyses indicated a strict correlation between the HBsAg secretion and HBV DNA level. The core antigen was detected in the cytoplasm of HC-T and was occasionally detected in the nucleus.These results revealed that at least 10 percent of HC-T cells were infected.The HC-T had the same HBV infectivity features as the freshly isolated primary hepatocytes.4. HBV infection of mice with chimeric HC-T. The HC-T cells exposed to HBV were transplanted to the liver of nude mice via caudal veins. HBV DNA and proteins were detected in the serum of the injected mice for 2 weeks by ELISA and Real-time PCR individually. The core antigen was detected in the cytoplasm of liver tissues.The results pave the way for mice model of other hepatotropic viruses, such as HCV.In conclusion, transgenic human hepatocytes were developed by transduction with the gene encoding the hTERT. The transgenic human primary hepatocytes had the same features as the human primary hepatocytes on the morphologic, phenotypic, molecular and functional levels.The most important is its unique susceptibility to HBV infection. The establishment of transgenic human primary hepatocytes provides the foundations to study new antiviral agents or vaccines. The transgenic human primary hepatocytes are very promising for many other applications especially in the development of types of antiviral drugs that interfere with early step of infection.