Up-regulated HHGF Promotes Human Bone Marrow Stem Cells To Develop Into Hepatocyte-like Cells

China has 9.3 million carriers of hepatitis B virus,and 2 million of them are Hepatitis patient,The incidence of Non-alcoholic liver disease and alcoholic liver disease have increased year by year . A number of liver damage factors have led to tissue dysplasia,liver function decompensated,Ascites, liver cancer, upper gastrointestinal bleeding and a series of serious complications.Drugs is difficult to treat,Liver transplantation is the most effective treatment of the end-stage liver disease at present, but trapped in lack of sources,greatly limited its clinical application. On account of the infinite value and multilineage differentiation potential, the bone Mesenchymal stem cells have become a hot study issue..Bone marrow stem cells have been shown to contribute to all three germ layers and can be differentiated into all cell types present in the body:Osteoblast,cardiomyocyte,Chondrocyte,Fat cells and liver cells.Use transplantating stem cells to treat liver diease in clinlic is widly accepted,and a number of experiments add medium with various growth factors to enhance the effect of stem cell transplantation in vivo or in vitro,but there is lack of gene transfected mesenchymal stem cells induced into liver-like cells in vitro experimental study.Researches have shown that HGF was one of the most important factor in initiating from bone marrow mesenchymal stem cells into liver cells.It is reasonable to assume that c-met played a important role in HGF's function. HGF and its receptor c-Met protein binding leads to c-Metβ-chain tyrosine Tyr-1234 and Tyr-1235 autophosphorylation,Thus activated receptor intrinsic tyrosine kinase activity . Result in increased cell division or differentiation, cell separation and increased exercise capacity, to fulfill its biological role. Objective:We cultured the bone marrow stem cell with Adherent methods, According to the gene transfer technology, the HGF gene was transfered into human bone marrow mesenchymal stem cells. G418 selected transfected cells after the hHGF gene was transfected into hBMSCs mediated by Lipofectamine 2000. The expression of hHGF was confirmed to be up-regulated in the pcDNA3.1⁽⁺⁾-hHGF transfected hBMSCs by confocal laser, RT-PCR and western blot respectively. Then the pcDNA3.1⁽⁺⁾-hHGF transfected hBMSCs were cultured with the serum of alcoholic liver cirrhosis patient for 7 and 14 days. Western blot was performed to detect the alteration ofα-fetoprotein(AFP) and albumin (ALB) expression. To investigate the effects of human hepatocyte growth factor(hHGF)gene on the human bone marrow stem cells (hBMSCs) developing into hepatocyte-like cells. The main results and conclusions are as follows:Method:1.Construction the recombinant pcDNA3.1⁽⁺⁾-hHGF,this step is maintainly used Molecular biology techniques,the HGF cDNA was ampified by poly-merase chain reaction(PCR),and then, XbalI and BamHI enzyme digested it and inserted into the enzyme sites. T4 ligase Connection them, Construction The recombinant vector.2.Adherent cultured and amplified the bone marrow stem cells, which was simple and easy to manipulate.and then select the Good growth cells to Transfection.3.The recombinant plasmid pcDNA3.1⁽⁺⁾-hHGF transfecting the huamn bone marrow stem cells mediated by lipfectamine 2000 , The expression of hHGF in transfected cells was detected by confocal laser, RT-PCR and western blot respectively.4.Then the pcDNA3.1⁽⁺⁾-hHGF transfected hBMSCs were cultured with the serum of alcoholic liver cirrhosis patient for 7 and 14 days. Western blot was performed to detect the alteration ofα-fetoprotein(AFP) and albumin (ALB) expression.Result:1.The hHGF cDNA was particularlly amplified,to reconstruct and analysis the plasmid by restriction endonuclease digestion and sequencing .the segment of hHGF gene was Absolutely correct. 2.Successfully cultured the cells,which was appeared Spindle and the fish-like growth, selected the good condition growth of human bone marrow mesenchymal stem cells for transfection.3.pcDNA3.1⁽⁺⁾-hHGF could transfected human bone marrow mesenchymal stem cells mediated by lipofectamine 2000.selected by G418, we effectively increase both the hHGF mRNA and protein Confirmed by confocal laser, RT-PCR and western blot ;4.After transfection, Induced human bone marrow mesenchymal stem cells gradually into epithelial-like cells, triangular or irregular in shape. Uninduced cells did not change significantly,The expression of AFP and ALB obviously increased in the pcDNA3.1⁽⁺⁾-hHGF transfected cells after co-cultured with the serum of alcoholic liver cirrhosis patient for 7 and 14 days.Conclusion:1.We amplified hHGF cDNA full-length sequence and successfully inserted into vector pcDNA3.1⁽⁺⁾;2.Adherent culture can nurture the human bone marrow mesenchymal stem cells,and it can Extensive expansion in vitro;3.pcDNA3.1⁽⁺⁾-hHGF could transfected human bone marrow mesenchymal stem cells mediated by lipofectamine 2000, hHGF gene in transfected cells can be successfully express the target protein.4. Alcoholic liver cirrhosis patient's serum could induce human bone marrow mesenchymal stem cells into liver-like cells, and hHGF gene expression in cells improve the ability of human bone marrow mesenchymal stem cells into hepatocyte-like cells.In a words, pcDNA3.1⁽⁺⁾-hHGF tranfected hBMSCs could apparently up-regulate the expression of HGF. Exogenous HGF gene could promote hBMSCs to induce into hepacyte-like cells, which may be a promising gene therapeutic method for terminal stage hepatopaths.