| Background and Aims:End-stage liver disease badly do harm to human health. While, liver transplantation is limited due to liver sources, costs and the side-effect of immunosuppressive agent and so on. The stem cell transplantation provided a better treatment for this kind of diseases. Bone marrow mesenchymal stem cells(BMSCs) is a kind of cells which have strong self-replication ability, multiple differentiation potentialities and low immunogenicity. It is expected to be a new seed cells which can be used to treat end-stage liver diseases. At present, the research proves BMSCs participate in damaged liver tissue repair, but the mechanism is not very clear, whether for most researchers believe that stem cell cross mesoderm differentiation. At present, there are some hospitals already use the stem cells to treat the end-stage liver disease, but transplantation efficiency problem such as transplant ways remains to be seen. Thus, in order to solve these problems, we use in-vitro induced experiment to validate BMSCs can differentiate into hepatocyte. We injected the BMSCs through portal vein and peripheral vein two different ways, to analyze the colonization and the liver function recovery status. The research results can provided experimental basis for clinical application.Method:1.We use density gradient centrifugation and adherent cell co-culture method isolated BMSCs, through continuous cell culture to gain pure BMSCs.2.Treated purified BMSCs by using hepatocyte growth factor(HGF) and basic fibroblast factor-4(FGF-4), then, We using immunocytochemical staining to detected the expression of liver cell surface markers, such as albumin(ALB), alpha fetoprotein(AFP), cytokeratin 18(CK18).3.Subcutaneous injection with CCl4 to the inside of rat limbs, transplanted the BMSCs which marked by green fluorescent protein(GFP) into the Cirrhotic Rats through tail vein and portal vein. And then, using the fluorescence microscope to observe the colonization situation of the BMSCs. At the same time, examine the liver function of different group rats.Results:1.Using density gradient centrifugation and adherent cell co-culture method, we can obtain the BMSCs in high purity and activity in vitro proliferation.2.After co-treated the rat bone marrow mesenchymal stem cell with HGF and FGF-4 for 28 days, we can detected the expression of the hepatocyte surface markers using immunocytochemistry. With the prolonged induction time, the expression of albumin and keratin 18 are increased, while the expression of AFP is gradually decreased. And BMSCs treated without HGF and FGF-4 did not expressed three proteins.3.HE and Masson staining confirm that the liver cirrhosis rat modeling success. We use the lentiviral carrying GFP to transfection the BMSCs. After transfection, the BMSCs can stable expression GFP. BMSCs after liver transplantation by portal vein and peripheral vein grafts 28 days, were scattered sections of green fluorescent protein positive cells, BMSCs can be planted in the liver after transplantation in vivo rat model; the liver function indicators such as albumin(ALB), Alanine transaminase (ALT), Aspertate Aminotransferase(AST) of transplantation groups were gradually improved with prolonged time. Comparing to control group, the differences were significant. But there are no significant improvement of liver function between tail vein group and portal vein group.Immunohistochemical detected that compared with the control group, the albumin expression in liver tissue of the experimental group was statistically significant; two experimental groups was not statistically different.Conclusion:1. BMSCs can be induced to hepatocyte-like cells in vitro.2. BMSCs transplantated through tail veins and portal veins can planted in the rats'damage livers. This method can improve liver function to some extent. 3. There was no significant difference of curative effect by BMSCs transplantated between through tail veins and portal veins. |
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