Establish Of Chimeric Mouse Model With HepaRG Cells Transfected Bcl-2 Gene Of Agonistic Apoptosis Ability Induced By Fas Antibody

The liver is one of the earliest organ selected for transplantation. Human hepatocytes transplantation is not only as a effective therapy technique, but also useful to establish animal model with allogeneic hepatocytes in understanding liver disease with virus infection.To establish an ideal animal model of mouse with chimeric human liver, human hepatocytes must be exist in the mouse for a long time with biologic function. There are three pacing factor for exogenous hepatocytes transplanted into rodent animal. First, without immunologic rejection. Second, damage recipient hepatocytes to form hepatic regeneration demand. Third, educe proliferative advantage of transplantation hepatocyte.With Bcl-2 gene of agonistic apoptosis ability induced by Fas antibody in vivo, we supposed it is very useful to built a mouse model with chimeric human liver.Fas antigen, CD95, is a surface protein (receptor). It belongs to the TNF (tumor necrosis factor)/NGF receptor family, which can mediate apoptosis when stimulated with agonistic anti-Fas antibody (Jo2 antibody) or Fas ligand. Bcl-2 is a one of plasmosins. It belongs to ever-growing family of protein which are of key point in apoptosis proceeding. It has been proved that the Jo2 antibody recognizes mouse Fas, and mouse hepatocytes is sensitivity to Jo2 antibody by inducing apoptosis. Repeated small dose of Jo2 antibody used in mouse did not appear sufficient to engage fas in extra-hepatic tissues, and result in liver large-area apoptosis, sometimes with fulminant hepatic failure and death. The present study demonstrated that Bcl-2 gene expression in allogeneic transplanted hepatocytes completely inhibited apoptosis by Jo2 antibody induced and thus improved survival and hepatocyte function of the donor cells. It provide a novel strategy to built an animal model of mouse with chimerical human liver with Bcl-2 gene expression resistant to fas-induced apoptosis and mouse recipients treated with Jo2 antibody.HepaRG cell line is from the non-tumoral region of a resected hepatitis C virus-associated hepatocellular carcinoma (HCC). It is a naturally immortalized human liver cell line with progenitor properties and bipotent differentiation inducible capability. Compared with other hepatoma cell lines, it manifests three important hepatic functional character. (1) a high proliferative ability retained a long-term stable expression of several liver-specific markers, including some CYP- associated activities; (2) differentiation potential toward biliary and hepatocytic lineage; and (3) the ability to display optimal metabolic functions associated with a unique susceptibility to HBV infection. Later studies demonstrated that, these bipotent progenitor cells have been found to repopulate uPA/SCID mouse damaged livers and exhibited a set of characteristic features that contributes to define the human hepatic bipotent progenitor signature in vivo. Moreover, HepaRG cells did not result in mouse oncogenicity when transplanted into nod mice.In this study, we will transplant HepaRG cells transfected Bcl-2 gene into severe combined immunodeficient (SCID) mouse using Jo2 antibody injection. This specific Jo2 antibody regimen appears to provide both the necessary"space"and sustained priming and proliferative signals necessary for liver regeneration by the engrafted donor hepatocytes. Meanwhile, Bcl-2-modified hepatocytes as donor diminishes apoptosis by Jo2 antibody and could be robust repopulation more than recipient mouse hepatocytes. Furthermore,model of xenogeneic hepatocyte transplantation in normal SCID lacking T and B lymphocytes lead not to immunologic rejection.Methods and Results1. Analyze the effects of Jo2 antibody on mouse hepatocytes and HepaRG cells in vitroTo investigate the effects of Jo2 antibody on apoptosis in primary cultured mouse hepatocytes and HepaRG cells, SCID mouse hepatocytes were isolated by using collagenase irrigation and HepaRG cells were cultured. Mouse hepatocytes and HepaRG cells were treated with Jo2 antibody (final concentration of 10μg/mL, 5μg/mL, 1μg/mL and 0.1μg/mL respectively ) under common growth condition.Treated with Jo2 antibody (5μg/mL) for 72 h and with Jo2 antibody (10μg/mL) for 48 h and 72 h, apoptosis of these three-groups of mouse hepatocytes were induced, however, HepaRG cells did not detected apoptosis signs induced by different concentration of Jo2 antibody. Mouse hepatocytes apoptosis were showed by cell loss of volume, cytoplasm condense, nucleus fixed and cell refractive index increased. Apoptotic body were found in mouse hepatocytes by Write staining. Inhibition ratio of three-groups of mouse hepatocytes were 52%,51% and 67% respectively measured with MTT assay. Detection of DNA ladder in an agarose gel after Jo2 antibody induced in these three-groups of mouse hepatocytes. Alanine transarninase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were not maked changes.2. HepaRG cells engraftment in SCID mice using Jo2 antibodyTo study effect of HepaRG cells engraftment in SCID mice using Jo2 antibody, acute liver failure was induced by Jo2 antibody (0.3mg/kg) treated intraperitoneally in SCID mice. Mice received 2×106 HepaRG cells engraftment within 24 hours after Jo2 mAb administered were divided into experimental group (n=10), and non-transplanted mice administered Jo2 antibody were divided into control group (n=10) respective randomLy. Nine transplanted mice were survival after 4 weeks and nine non-transplanted mice were dead successively within 3 days. Compared to control group, survival time was prolonged significantly and serum liver enzyme levels was reduced significantly of experimental group( P < 0.01). HepaRG cells engraftment protected mice from Jo2 antibody mediated liver hemorrhage and hepatocyte apoptosis in contrast to matched non-engrafted mice. After 4 weeks post-engraftment, human albumin, CK18 and specific human Hep Par 1 were detected by immunohistochemistry staining, and human albumin were also detected by immunofluorescence staining. Though HepaRG cells are able to exist in damage mouse liver, the repopulation potentiality of HepaRG cells in the mouse is much limited.3. Construction and expression identification of stable-transfected Bcl-2 gene into HepaRG cellsThough HepaRG cells did not detected apoptosis signs induced by Jo2 antibody in vitro, transplanted HepaRG cells survived and regenerate with limited for absence of agonistic apoptosis ability induced by Jo2 antibody in vivo. So, we construct stable transfection HepaRG cell line of the anti-apoptotic gene Bcl-2 inhibits Jo2 antibody induced cell injuries. Cells were collected and grouped as following: cells transfected with plasmid of pSFFV-neo Bcl-2, the stably transfected cells were double selected by culturing in medium containing G418 and anti human Fas antibody.To identify expression of stable-transfected Bcl-2 gene into HepaRG cells, Bcl-2 protein was found in different assays. Stable-transfected Bcl-2 HepaRG cells were not found morphological marked changes. Human albumin was detected in transfected cells by immunohistochemistry staining. Bcl-2 mRNA were measured by reverse transcription- polymerase chain reaction (RT-PCR). Bcl-2 protein were also found by Western bolt. Bcl-2 protein did not present in non-transfected HepaRG cells or transfected empty plasmid HepaRG cells.4. Establish of chimeric mouse model with Bcl-2-transfected HepaRG cellsRepopulation of SCID mouse liver with Bcl-2 transfected HepaRG cells was studied following induction of Jo2 antibody. Bcl-2/HepaRG-mouse chimeric liver, that is, SCID mice were transplanted with 2×106 Bcl-2 transfected HepaRG cells intrasplenically and treated once weekly for 10 weeks with 0.2mg/kg Jo2 antibody intraperitoneally consistently (n=43). HepaRG-mouse chimeric liver, in other words, mice were transplanted with non- transfected HepaRG cells and treated with Jo2 antibody (n=25). Mice were transplanted with non-transfected HepaRG cells and non-treated with Jo2 antibody regarded as control group (n=14).Three groups of mice were survival to 24 weeks. Each group of several mice were euthanized at various time points, ranging from 2 to 24 week post-engraftment. Immunohistochemistry and immunofluorescence staining confirmed the expression of human Bcl-2 in Bcl-2/HepaRG-mouse chimeric liver. From 2 week posttransplantation Bcl-2 expression gradually inceased. After it reached peak concentration at 12 week posttransplantation, then, decreased slowly up to undetectable after 24 week posttransplantation.Mice liver of three groups were able to detected human albumin and cytokeratin 18 by immunohistochemistry, and immunofluorescence staining for human albumin and Ki67. From 2 weeks after transplantation, in Bcl-2/HepaRG -mouse chimeric liver, human albumin,cytokeratin 18 and Ki67 were synchronously detectable up to24 weeks after transplantation, but up to 20 weeks for HepaRG-mouse chimeric liver and up to 12 weeks for control group only. High-peak of these hepatic feature marker appearanced at 16 weeks for Bcl-2/HepaRG-mouse chimeric liver, 12 weeks for HepaRG-mouse chimeric liver and 8 weeks for control group. After meticulous analyses of sections of chimeric mouse liver for human specific markers, we estimate 22%, 15% and 8% of them are of human origin respectively in Bcl-2/HepaRG-mouse chimeric liver, HepaRG-mouse chimeric liver and control group, even though it is practically difficult to assess the exactly level of the contribution of human hepatocytes in the transplanted liver. Meanwhile, RT-PCR for human albumin mRNA in mice liver were detected respectively from 4 to 24 weeks in Bcl-2/HepaRG-mouse chimeric liver, from 4 to 20 weeks in HepaRG-mouse chimeric liver and from 4 to 12 weeks in control group after transplantation, Western blot assay for human albumin were turned out at the same time in these three group mice serum. The quantity of human serum albumin by enzyme linked immunosorbent assay(ELISA) in mice serum at the same time, maxlmum was 0.101μg/mL in Bcl-2/HepaRG-mouse chimeric liver at 16 weeks, 0.042μg/mL in Bcl-2/HepaRG -mouse chimeric liver at 12 weeks, and 0.022μg/mL in control group at 8 weeks after transplantation.Conclusions1. Mouse hepatocytes could be induced apoptosis by Jo2 antibody in vitro, however, HepaRG cells did not detected apoptosis signs induced by Jo2 antibody.2. HepaRG cells transplantation can improve survival with unchanged histology of model mice and protect fulminant hepatic failure by Jo2 antibody treated intraperitoneally. However, transplanted HepaRG cells survived and regenerate in mouse liver are limited for absence of agonistic apoptosis ability induced by Jo2 antibody in vivo.3. HepaRG cells stable-transfected Bcl-2 gene is no diference with HepaRG cells in appearance and biological function, and has been shown expression of Bcl-2 protein.4. With Bcl-2 gene of agonistic apoptosis ability induced by Jo2 antibody in vivo, Bcl-2 gene could be found in the SCID chimeric mouse liver, and survival of functional HepaRG cells could be detected for 24 week after transplantation either. These data indicated that HepaRG-mouse chimeric liver model is successful. For Bcl-2 gene transfected, HepaRG cells has longer survival, more quantity and higher amount of human albumin expression in Bcl-2/HepaRG-mouse chimeric liver model than those in HepaRG-mouse chimeric liver model.