A Preliminary Research On Differentiation Mechanism Of Mesenchymal Stem Cell Into Insulin-secreting Cells In Pancreas Microenvironment

Objective To object the therapeutic effect and the cell distrubution after bone marrow mesenchymal stem cells(BMSCs) transplanted into pancreas microenvironment of diabetic rats model,and to investigate the mechanism of BMSCs differentiated into insulin-secreting cells.Methods BMSCs of allogeneic male SD rat were isolated from bone marrow,purified and transfected with enhanced green fluorescent protein(EGFP) for tracing,administered them to diabetic rats by injecting into subcapsular pancreas at multiple points.Blood glucose levels, blood insulin levels and blood C-peptide levels were monitored.The distribution of BMSCs were detected by EGFP.The expression of PDX-1,ngn3,nestin,Nkx2.2,Pax 4 in these EGFP positive pancreas were analyzed by Real-time quantitative RT-PCR at 1,2,3,5,8 and 12 weeks..EGFP and insulin double-positive cells were detected by immunofluorescence.EGFP and PDX-1 mRNA double-positive cells were detected by fluorescence in situ hybridization(FISH) at 8 weeks after transplantation.FCM were performed to analyse DNA ploidy whether there are fusion of BMSCs with islet cells.Resalts Blood glucose reduced continuely 6 days after transplantation,approached normal rats level in 24 days after transplantation(150±42.0mg/dL ),insulin and C-peptide significantly increase than that of normal rats in 14 days after transplantation,a high level stabilized to 56 days after transplantation(1.0±0.2μg/L,1.2±0.3 nmol/L ).Expression of EGFP in cells receptor rats tissue stabely up to 8 weeks after transplantation.EGFP labled cells were distributed in islets(12.46%),blood vessel(4.4%),(9.24%),duct(3.21%),local acinus and tissue space(79.93%). The double-positive of EGFP and insulin is 5.16%in cells.,The double-positive of EGFP and PDX-1 mRNA is 0.96%in cells.The exprssion od PDX1,ngn3,nestin,Nkx2.2 and pax4 up-regulation to peak 5 weeks after transplation,then down-regulated,ngn3,nestin,pax4 didn't detected in 12 weeks after transplation.These were diploid and tetraploid but no polyploid and aneuploid in BMSCs transplated pancreas.Conclusions Transplantation of BMSCs into pancreas effectively reversed hyperglycemia of experimental diabetes rat,increased blood insulin level and C-peptide level.Those transplanted cells can redistributed in pancreas,and differentiated into insulin-secreting cells within pancreatic microenvironment.The mark genes of[3-cells' developing were expressed during differentiation.There were no fusion of BMSCs and tissue cells.