| Hepatitis B is the most common and serious liver disease, especially in developing countries. Although HBV pathogenesis has been extensively investigated and we have known much about the relationship between HBV infection and the immune system, the proteomic alteration of hepatocytes during HBV chronic infection is still unclear. More and more evidence indicates that HBV proteins regulate the expressions of hepatocyte proteins and combinations of HBV proteins have different effects. On the other hand, differentially expressed proteins caused by HBV infection might change the immunological recognition in the organism and cause the formation of autoantibodies. Our study is to investigate how HBV proteins influence the expressions of hepatocyte proteins and to compare the relation and differences of hepatocyte proteomic alteration in the existence of different HBV proteins. Meanwhile, 2-DE and immunoblot are used to identify autoantibodies in serum of HBV-Tg mice and HBV patients.First, using the purified hepatocytes, we compared the protein profiles by two-dimensional electrophoresis (2-DE) and Liquid Chromatography/Mass Spectrometry (LC-MS) between HBV transgenic and corresponding background mice. In this study, twenty seven altered proteins were identified in hepatocytes from HBV-transgenic mice, among which seventeen proteins were upregulated while ten were downregulated. Bio informatics analysis showed that thirteen proteins were involved in mitochondrion metabolism pathway including tricarboxylic acid cycle (TCA) and oxidative response; four proteins (SELENBP, SCP2, RGN and PRDX1) were also dramatically changed in liver samples from HBV-infected patients. Important genes (gpx, sod, ogg et al.) correlated to oxidative damage were upregulated in the liver of HBV-Tg mice. Reactive oxygen species production was significantly increased while ATP production was decreased in liver mitochondria from HBV-Tg mice. Moreover, hepatocytes of HBV-Tg mice were more sensitive to hydrogen peroxide-induced cell death than wild type control.Meanwhile, we also have compared hepatocyte proteomic alteration in the existence of different HBV proteins. First, the proteomics profiling of HBs-Tg mice, partial gene HBV-transgenic mice, was compared with that of wide type mice with the same genetic background. LC-MS analysis identified twenty differentially expressed proteins in hepatocytes from HBs-Tg mice, including sixteen upregulated proteins and four downregulated ones. We found that among the twenty altered proteins identified in HBs-Tg mice, sixteen were consistent with those of HBV-Tg mice.2D-western blotting analysis indicated autoantibodies in serum of both HBV-Tg mice and HBs-Tg mice. Eight hepatocyte proteins were found to react with sera of HBV-transgenic mice, among which two proteins were upregulated in HBV-Tg mice. We further investigated the subcellular localization of those self-reactive proteins with GO analysis and found that seven of out eight were located in hepatocyte mitochondria. Four hepatocyte proteins were found to react with sera of HBs-transgenic mice but not that of back ground mice respectively, 3 of which overlapped with that of HBV-Tg mice. In the clinical experiments, we have identified nine autoantibodies successfully, 3 of which (ECSH1, ALB and GLUD) were consistence with that of HBV transgenic mice, anti-GLUD antibody had a high reoccurrence (5/30) revealed the potential of GLUD as a clinical target of diagnosis and therapy.Our study compared the proteome differences between HBV infected mammalian hepatocytes and wild type hepatocytes and further verified the bioinformatics analysis results. Meanwhile, we also analyzed the seroproteome of HBV-Tg mice and HBV infected patience. We believe this study is the first proteomic and seroproteome analysis of HBV infected mammalian hepatocyte and provides theoretical foundation for the mechanism of how HBV proteins affect hepatocyte protein expression and the organisms' response to those changes. |
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