| BackgroundFulminant hepatic failure (FHF) has very high mortality rates approaching70-80%. Considering the potential of the liver to regenerate, Liver support system has been considered to the alternative therapy for liver failure in clinical trial as a bridge to orthotropic liver transplantation(OLT) or avoiding liver transplantation. To date, several kind of bioartificial liver (BAL) using primary porcine hepatocyte, primary human hepatocytes and C3A liver tumor cells have been submitted for clinical trials. However, no BAL has been approved by government for clinical treatment, because suitable sources of liver cells are not available. So, it is very urgent and important to find new type of hepatocytes for application in BAL as the biological component of BAL.The immortalized human hepatocytes with indefinitive expansion in vitro and allogenic cells are an ideal suitable for application in BAL. However, its functionality and differentiation is lower than that in primary human hepatocytes. Liver progenitor cells can be expanded and differentiated into functional hepatocytes that are urgently required as potentially alternatives to hepatocytes.Cell-cell interactions such as hepatocytes and stellate cells play a very important role in the stability of liver function of primary hepatocytes. However, human hepatic stellate cells (HSC) are terminally differentiated liver cells, which have a limited proliferation and do not passage in vitro for long term time. Moreover, isolation of HSC is extremely time-consuming and laborious works, their yields are commonly even lower.Here, we aimed to establish an immortalized human hepatic stellate cells line, and to evaluate the effects of co-culture with the immortalized human hepatic stellate cells on hepaocyte functions of immortalized human hepatocytes and differentiation of liver progenitor cells into functional hepatocytes.Part I Establishment and characterization of an immortalized human hepatic stellate cells lineObjective:To establish an immortalized human hepatic stellate cells Line for either co-culture of hepatcotyes and human hepatic stellate cells or co-culture of liver progenitor cells with human hepatic stellate cells in vitro as an ideal cells line.Methods:Primary human Hepatic stellate cells were transfected with recombinant retrovirus containing simian virus40large T antigen (SV40LT). Subsequently, the characteristics of immortalized human hepatic stellate cells was characterized by reverse transcription polymerase chain reactions (RT-PCR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescent cytochemistry staining, and so on.Results:An immortalized human hepatic stellate cells Line, HSC-Li, was harvested after infection of primary human hepatic stellate cells with recombinant retrovirus containing SV40LT. Under phase contrast microscope and electronic microscope, the immortalized human hepatic stellate cells showed classical appearance of hepatic stellate cells. The mRNA expression of human Collagen type I, Collagen type Ⅱ、HGF and SV40LT was observed by RT-PCR in HSC-Li cells. The results of immunofluorescent cytochemistry staining showed that HSC-Li cells expressed α-smooth muscle actin (a-SMA), Vimentin, and glial fibrillary acid protein (GFAP). The cytokines such as HGF, VEGF, IL-6, and TGF-betal were determined in the culture supernatant of HSC-Li cells by ELIS A and protein array. Conclusions:The immortalized human hepatic stellate cells, HSC-Li cells, have the biological and functional characteristics of human hepatic stellate cells, which may be an ideal cells line for either co-culture of hepatocytes with hepatic stellate cells or co-culture of liver progenitor cells with hepatic stellate cells in vitro. Part II Effects of co-culture with immortalized human hepatic stellate cells on the hepatocyte function of immortalization human hepatocytesObjective:To evaluate the effects of co-culture with immortalized human hepatic stellate cells on hepaocyte function of immortalized human hepatocytes for application in bioartificial liver.Methods:Immortalized human Hepatocytes were established by transfection of primary human hepatocytes with recombinant retrovirus containing SV40LT. Characteristics of immortalized human hepatocytes were evaluated by analysis of gene expression and functional characteristics in vitro. After co-culture with the HSC-Li cells employing mixed co-culture and separated co-culture, the mRNA expression of metabolizing enzymes and CYP1A2activity of immortalized human hepatocytes was evaluated.Results:An immortalized human hepatocytes cell line, HepLi5, was successfully established. The expression of SV40LT in HepLi5cells could be detected by RT-PCR and Western blot. The mRNA expression of liver-enriched genes and human cytochrome P450was detectable by RT-PCR in HepLi5cells.When HepLi5cells were co-cultured with HSC-Li cells in either separated co-culture or mixed co-culture, the CYP1A2activity was significantly enhanced. The mRNA expression levels of metabolizing enzymes such as CYP3A5, CYP2E1and UGT2b7of HepLi5cells were significantly improved in both separated co-culture and mixed co-culture with HSC-Li cells. Conclusions:we have successfully established an immortalized human hepatocytes cell line, HepLi5. Our results suggested that co-culture of HepLi5cells with HSC-Li cells significantly improved the gene expression and functional characteristics of HepLi5cells. The hepatocyte function of immortalized human hepatocytes could be enhanced in separated co-culture and in mixed co-culture with HSC-Li cells, which may be greatly helpful for use in BAL in future. Part Ⅲ Effects of co-culture with immortalized human hepatic stellate cells on the differentiation of liver progenitor cells into functional hepatocytesObjective:To evaluate the effects of co-culture with the immortalized human hepatic stellate cells on the differentiation of liver progenitor cells into functional hepatocytes in vitro.Methods:liver progenitor cells, BMEL-TAT, and the immortalized human hepatic stellate cells, HSC-Li cells, were used in this study. The separated co-culture of BMEL-TAT with HSC-Li cells was established for analyzing the effects of co-culture with immortalized human hepatic stellate cells on the differentiation of liver progenitor cells into functional hepatocytes. The cell morphology of BMEL-TAT cells was observed by light microscope and laser confocal microscope. The mRNA expression of ALB, CK18, TAT and G6P in the BMEL-TAT cells was detected by RT-PCR after co-culture with HSC-Li cells. The CYP1A2activity, Dil-LDL uptake, and urea secretion were also determined in both BMEL-TAT control and after21days co-culture of BMEL-TAT, etc. The BMEL-TAT cultured on normal medium served as control.Results:co-culture with HSC-Li cells significantly enhanced the differentiation of liver progenitor cells into functional hepatocytes. The cell morphology of BMEL-TAT cells was changed to hepatocyte-like cells from days3to days21after co-culture with HSC-Li cells. The mRNA expression of ALB, CK18, TAT and G6P in the BMEL-TAT cells was significantly improved by RT-PCR after co-culture with HSC-Li cells. The results of immunofuorescent cytochemistry staining showed the expression of ALB, CK18was significantly enhanced in BMEL-TAT after21days co-culture with HSC-Li cells, whereas the expression of CK19and AFP apparently decreased. The CYP1A2activity, Dil-LDL uptake, urea secretion was also increased significantly after21days co-culture of BMEL-TAT with HSC-Li cells. X-gal staining and PAS staining in BMEL-TAT after21days co-culture with HSC-Li cells were apparently positive, whereas the BMEL-TAT control was negative.Conclusion:Separated co-culture with HSC-Li cells could successfully induce the differentiation of liver progenitor cells into functional hepatocytes, which may be very important for generation of functional hepatocytes from liver progenitor cells for use in BAL in future. |
PDF Full Text Request