| Objective: To investigate the effect of bone marrow mesenchymal stem cells modified by human hepatocyte growth factor gene.Methods:⑴Bone marrow mesenchymal stem cells(BMSCs) from the bone marrow were isolated by Percoll density gradient centrifugation and identitied by flow cytometry; The capacity of multiple differentiation were identitied by adipogenic and osteogenic induction.⑵Human hepatocyte growth factor (hHGF) gene was amplified by PCR method, and constructed recombinant plasmid encoding hHGF gene via gene recombination technology. Recombinant vector was identified by restriction enzyme digestion and DNA sequencing.⑶BMSCs were transfected with the recombinant plasmid by electroporation. The transfection efficiency were detected by inverted fluorescence microscope. The expression of hHGF gene in the transfected BMSCs were detected by RT-PCR and Western Blot. The proliferation of the transfected BMSCs were evaluated by MTT method.⑷BMSCs embellished with hHGF gene were induced by the serum of alcohol-hepatocirrhosis patient in vitro, the expression ofα-fetoprotein(AFP) and albumin (ALB) were detected via Western Blot at 14d. Result:⑴The flow cytometry confirmed that the cells we obtained were BMSCs. Adipogenic and Osteogenic induction confirmed that the cells have the characteristics of multipotent differentiation.⑵The target gene hHGF amplified by PCR from the vector Pblast2-hHGF,was identified by sequencing ,which proved that the hHGF gene was consisted of 2184 nucleotides and the completely same with the sequence published on Genebank. The recombinant plasmid was verified correctly by enzyme digestion as well as sequence analysis.⑶The green fluorescence were observed through fluorescence microscope after 48 h. The transfection efficiency was 38.2±2.1%, RT-PCR and WesternBlot demonstrate the expression of the hHGF in BMSCs embellished with hHGF gene. The results of MTT method showed that proliferation of hHGF-transfected BMSCs were increased.⑷After induced by the serum of alcohol-hepatocirrhosis patient about 14 days , BMSCs became into a spindle-shaped, irregular shaped or polygonal cells with the induction time, the number gradually increased and the morphology looked like hepatocyte. Western Blot showed that the expression of AFP and ALB in BMSCs modified by hHGF gene were higher than the control groups.Conclusion: BMSCs modified by hHGF gene could promote their differentiation into hepatocyte-like cells by induced with the serum of alcohol-hepatocirrhosis patient in vitro . |
