1. Cytokines Induced Hepatic Progenitor Cells Differentiation In Vitro 2. Construction Of Prokaryotic And Adenoviral Expression System Of HCV F Protein

Objective : Orthotopic liver transplantation, the only effective therapeutic option for end-stage liver disease, is mainly limited by the availability of donor organs. Recently, hepatocyte transplantation has developed into a feasible alternative to whole-organ transplantation, but isolation of sufficient transplantable hepatocytes is restricted by the small number of marginal donor organs allocated for this purpose.Cell-based therapy is a potential alternative to orthotopic liver transplantation. Stem cells are cells endowed with the unique property of perpetuating themselves through self-renewal while maintaining pluripotency, the ability to differentiate into all types of cells. The search for novel cell resources has prompted investigations into generating hepatocytes from stem cells of extrahepatic origin, based on their ready availability and unrestricted potential to propagate and differentiate .Embryonic liver stem cells are progenitor cells, which can only differentiate into hepatic cells and bile duct epithelial cells. The intermediate state is an essential process of differentiation from stem cells to mature liver cells. Studies on embryonic liver stem cells are required to clarify the mechanism of liver development,promote stem cells to differentiate into mature liver cells, but not tumor cells, for improving liver stem cells-based approach for liver regenerative therapy.We plan to obtain immortalized progenitor cells for further differentiation study and search for the optimal approach for hepatocyte-directed differentiation of hepatic progenitor cells. Moreover, we will investigate the molecular mechanism of the hepatic differentiation, and explore the effect of HGF, LIF, BMP2 or BMP9 in the differentiation process of hepatic progenitor cells.Methods: We transduced the retrovirus vetor SSR#69, which containing SV40T into hepatic progenitor cells and obtained the immortalized hepatic progenitor monoclone. In vitro differentiation assay, we add recombinant adnovirus which containing human LIF, BMP2 or BMP9 gene into hepatic progenitor cells. Cell state of maturation and differtiation was examined by PAS sraining and ICG uptake method at day 4, 7 and 10. Also, production of Albumin (Alb) was measured by luciferase activity at day 5, 7 and 10.Results:We developed immortalized method for hepatic progenitor cells and obtained four representative stable lines, named HP13-6, 13-19, 14-2 and 14-19 respectively. Among them, HP13-6 and 14-19 displayed epithelial-like features under microscope, and the expressions of hepatic progenitor early markers in these two lines are obvious. PAS staining assay revealed that BMP2 and BMP9 enhanced Alb expression most obviously at day 7. In details, the percentage of positive cells was 30% and 45% respectively. Meanwhile , ICG uptake examination also showed that HP14-19 has some functions of mature hepatocytes by addition of BMP2 or BMP9. Luciferase activity indicated that BMP2 induced ALB-Luc activity most significantly. That is, compared with negative control, Alb expression was nearly upregulated 7 folds at day10, and LIF, BMP9 or HGF treatment enhanced Alb expression by 2-5 folds. For HP13-6 cell line, BMP9 treatment induced Alb expression up to 10 folds than that of control cells. Also, there are less inductive activities in LIF (8 folds), BMP2 (7 folds) and HGF (6 folds) treated group.Conclusion:We obtained immortalized fetal hepatic progenitor cells, which can be induced and differtiated into hepatocyte-like cells. Objective : Hepatitis C virus (HCV) is a worldwide pandemic, chronically affecting over 180 million people worldwide. It is a major risk factor for the development of hepatocellular carcinoma (HCC) and there is increasing experimental evidence to suggest that the virus plays a direct role in neoplastic transformation. Current estimates suggest 2–5% of patients with liver cirrhosis due to HCV develop HCC annually.There is no specific vaccine is currently available to prevent HCV infection. The standard anti-viral therapy is using pegylated interferon (IFN) in combination with ribavirin, which has yielded a sustained virological inhibition.But the long-term effects are still unknown . Hepatitis C virus (HCV) F protein is a newly discovered HCV gene product that is expressed by translational ribosomal frameshift. Little is known about the biological properties of this protein. The functional significance of F protein is presently unknown. Whether F and core proteins have both shared and distinct functions in the mechanism of HCC? Whether F protein has a role in the growth and development of transformed cells? These questions are not solved now.We want to construct a prokaryotic expression plasmid containing HCV F gene, and purify the recombinant fusion protein in E.coli system. And to construct an adenovirus vector containing F gene for further biological study.Methods: F gene of Hepatitis C virus was amplified by PCR method from plasmid H/FL (containing full length cDNA sequence of HCV 1a subtype), cloned into pET32a(+) vector, and then transformed into E.coli JM109. After identified by restriction digestion and DNA sequencing, recombinant plasmid was transformed into E.coli BL21 and induced with IPTG. The fusion protein trxA-F was further confirmed by Western blot analysis and purified by affinity chromatography method. Recombinant shuttle plasmid pAdTrack-TO4-HCVF was linearized and transformed into Escherichia coli BJ5183 carrying backbone plasmid pAdeasy-1to obtain adenovirus plasmid through homologous recombination .After lineralized by PacⅠenzyme, the recombinant adenoviral plasmid was transfected into HEK293 cells to package into adenoviral particles.Results: Restriction digestion and PCR screening shows that HCV F gene was cloned into pET32a(+) successfully. After BL21 was transformed with recombinant vector pET32a(+)–HCVF and induced with 0.5mM IPTG, a 35.4KDa protein band was found by SDS-PAGE, and this recombinant protein showed a highly specific and strong reaction with anti-His monoclonal antibody by Western blot analysis. For the construction of Adenoviral vector, HCV F gene was cloned into recombinant shuttle plasmid successfully.Conclusion: The prokaryotic and adenoviral expression plasmid pET32a(+)-HCVF was successfully constructed. After several cycles of amplification freezing and thaw high-titer Adenorival F was obtained.