Transplantation of bone marrow mesenchymal stem cells harboring fusing gene of enhanced green fluorescent protein and human insulin gene in treatment of diabetic mice / 第二军医大学学报

ABSTRACT

Objective:To construct a retroviral vector carrying both enhanced green fluorescent protein(EGFP) and human insulin gene for transfecting the bone marrow mesenchymal stem cells (BMSCs),and to observe the treatment effect of transfected BMSCs after transplanted into diabetes mice.Methods:Two gene segments of insulin-IRES-EGFP and IRES-EGFP were obtained by overlap extension PCR technology,and then the 2 segments were cloned into retroviral vector (pMSCV).The vectors were used to transfect BMSCs after packaged with PT67 cells.The expression of EGFP was observed by inverted fluorescence microscope and the expression of insulin gene was examined by RT-PCR in the infected BMSCs.The transfected BMSCs with 2 viruses were transplanted into diabetic mice separately.The blood glucose levels and body weights of mice were examined in 2 groups,and the results were compared with those of normal control group.Results:The recombinant retroviral vectors pMSCV-insulin-IRES-EGFP and pMSCV-IRES-EGFP were successfully constructed.The BMSCs infected by both vector stably gave out green fluorescence under microscope; the cells infected with pMSCV-insulin-IRES-EGFP also stably expressed human insulin gene.The blood glucose level of the mice transplanted with BMSCs transfected with pMSCV-insulin-IRES-EGFP was significantly lower than that transplanted with BMSCs transfected with pMSCV-IRES-EFGP (P