Effect Of Ethanol-induced Oxidative Stress And HBV Replication On Proteasome Of Hepatoma Cells

Hepatitis B virus(HBV) as a hepatotropic,noncytopathic virus,primarily infects hepatocytes and causes a series of liver diseases such as acute and chronic hepatitis,liver cirrhosis and hepatocellular carcinoma, which are among the most important human health problems worldwide.Immune response to viral antigens plays a crucial role in the pathogenesis of hepatitis B viral infections. However, when clonal expansion of cytotoxic T-lymphocytes (CTLs) is established, the next important restriction for elimination of infected cells is the availability of peptide-major histocompatibility complex (MHC) classâ… complexes, which are recognized by CTLs on the surface of target cells (hepatocytes). With the new recognition that the synergistic action of ethanol with HCV results in the suppression of MHC classâ… -restricted antigen presentation. Evidence for direct interaction with proteasome subunits has been obtained for the HBV protein to reduced MHC classâ… -restricted antigen presentation.In this study,we usede transfected cell line HepG2.2.15 and its parental cell Line HepG2 to research the effect of ethanol-induced oxidative stress and HBV replication on proteasome.Cells were stimulated by ethanol with gradient concentration(1.5%-8%) for 16 hours. The assay of AST release from the cultured cells was used to evaluate the cytotoxicity of ethanol. The inhibition rate of cells was determined by MTT method. Western Blot was utilized to detecte the expression of LMP2. Our major findings are shown as followe:HepG2.2.15 was more sensitive to 3% and 5% ethanol induced cell injury. The expressions of LMP2 were much lower in HepG2.2.15 cells compared with HepG2 cells with or without 1.5% ethanol exposure.Our study demonstrated ethanol and HBV down-regulated the expression of immunoproteasome subunit LMP2 in hepatoma cells.We deduce HBV replication and Ethanol-induced oxidative stress suppress the generation of peptides for MHC classâ… -restricted antigen presentation. AIM:The purpose of this study was to search a simplified, inexpensive method in isolating HBV-transgenic mice hepatocytes,and the primary hepatocytes of HBV-transgenic mice was observed. To explore the potency of HBV-transgenic mice hepatocytes as a vitro model in anti-HBV drugs.METHOAD:Mice hepatocytes were isolated by modified two steps collagenase perfusion,and then purificated by 40% Percoll.The viability of cultured hepatocytes was assessed by trypan blue exclusion. The morphologic change of cultured hepatocytes was observed, and the concentrations of albumin, urea,lactate dehydrogenase (LDH),HBS-Ag,HBe-Ag and HBVDNA in the supernatant collected from different cultural period of better isolated system were examined. Primary hepatocytes were treated with Bay-4109 to evaluate their pharmacodynamics and mechanism of action.RESULT:Hepatocytes obtained by modified two steps collagenase perfusion were intact and had a prosperous viability and an active function. The fluctuated changes of LDH leakage, albumin synthesis and urea levelwere displayed in one week, and the lesser LDH leakage and higher concentrations of albumin and urea were observed on the third day. The contents of HBS-Ag secretion mantained higher level from 1th to 7th day, HBe-Ag and HBVDNA decreased gradually over time in the period of culture. The HBS-Ag and HBe-Ag release from the cultrued cells decreased after Bay-4109 treatment.CONCLUSION:It is concluded that the collagenase digestion method is feasible for isolation of HBV-transgenic mice hepatocytes. Primary hepatocytes have an potentiality as a vitro model in anti-HBV drugs.