The Research Of BMSCS Used To Diabetes After Induced Into Islet-like Cells

[Objective]:Get bone marrow mesenchymal stem cells in vitro,cultured and amplified.Under the role of the inducer differentiate into islet cells at different stages.transplanted The various stages of the cell into rabbits model of diabetes.Observe the different stages of cell homing and differences in the repair capacity of the pancreas.[Methods]:Based on characteristics of BMSCs relatively low density and easy adherent growth,Isolated rabbit BMSCs by density gradient centrifugation and adherent culture combining.after amplification in vitro,cutured and amplified with DMEM containing10%fetal bovine serum (FBS),Secure a steady growth of bone marrow mesenchymal stem cells,And identified of bone marrow mesenchymal stem cells by morphological characteristics, surface markers.Experimental animals using a3-month-old New Zealand white rabbits, weighs about2.5kg.Forty rabbits were randomly divided into five groups,BMSCs group、7-day group、14-day group、21-day group and modeled group.We established rabbit Diabetes model,After successfully modeled, GFP-cells were injected into Diabetes model with3x106cells/kg through the ear vein,Then observed for glucose and body weight changes.Six weeks later, We maked tissue sections and observed efficacy different of ever stage BMSCs repair pancreatic after BMSCs’s transplantion.[Results]:①MSCs primary cells obtained by density gradient centrifugation, Culture4d that colony can be formed by BMSCs,About6-7d, cell density in colony and morphology was short spindle,Cells were digested with0.25%trypsin terminated with medium containing serum and subcultured,After passage,the cell growth is faster than the original generation,about5days fused to the85%-90%,spread to the three generations was the pure BMSCs confirmed by flow cytometry compared,Lentiviral GFP gene transfection does not affect the normal cell growth and differentiation,By flow cytometry cell surface markers suggest that MSC generation expression of CD34, CD45and not expressed CD73, CD105.The results show that we isolated and cultured BMSCs was cell populations with homogeneous cell indicates.②On the basis of successfully isolated and cultured BMSCs,We used two-step method for the induced BMSCs directed into insulin-secreting cells.Take well-grown3rd generation BMSCs,Seeded in6-well plates at a density of2×106cells/ml,added to each well2m1L-DMEM medium contains lOμg/ml basic fibroblast growth factor(bFGF),10μg/ml epidermal growth factor(EGF)and2%B27,Cultured for6days and removed the old culture liquid,washed three times with D-Hank’s solution,and then added H-DMED medium contains10μg/ml hepatocyte growth factor(HGF),10μg/ml beta cells regulate hormone(B-cellulin),10μg/ml activinA,10mmlo/ml of nicotinamide and2%B27,cultured for another6days, a slight change in cell morphology,most cells were round and a few were elongated spindle,some appear small cell clusters.approximately14d majority of cells clustered together and like islet cell in the struture.further Western blot detection and insulin immunohistochemical staining showed that the cell induced can synthesis and expression of insulin.our disign,thereby it’s provide a source of cells for further cells autologous transplantation.③To establish the diabetes animal model is a basic conditions of cell transplantation,we use New Zealand white rabbits as our experimental animals and intraperitoneal injection of streptozotocin method to selective destruction of pancreatic B cells,we successfully established of diabete animal model,After administration, the animals appear "little more than three" symptoms,fasting blood glucose showed that blood sugar fluctuations in3day injection.blood sugar stabilized and significantly higher after3days later, more than16.7mmol/L,compared before is significant difference(p<0.01),urine is always=+++and the weight gradually increses.experimental animals did not fall off, all included in the experiment.④The various stages of induced cell transplantated into animals,six weeks later,we killed the rabbit model and maked pancreas paraffin sections、HE staining,the results showed that14-days group was significant than model control group(p<0.01),14-day group was significant compare to BMSCs group,7-day group,21-day group(p<0.05),tissue morphology showed that cell homing and graft survival well,cells are able to aggregate to form cell clusters,immunohistochemical were positive,The blood glucose value is reduced,confirmed that the transplanted cells can survive and insulin secretion in the pancreas,the recipient animals return to normal blood sugar levels.[Conclusion]:The experiment successfully obtained the BMSCs、cultured in vitro、 amplified and identified BMSCs,Optimized conditions of inducing to differentiate into insulin-secreting cells in vitro,Successfully set up a stable and repeatable diabetic animal models,The evaluation and mechanisms of BMSCs differentiation in the different stages of cell transplantation in the treatment of pancreatic damage repair the curative effect,BMSCs transplantation group, induced group7days,21days of induction group, the decline in blood glucose levels after transplantation, compared with BMSCs induced14days HE staining showed infiltration of inflammatory cells, deeply stained large nuclei, small particles of endocrine.But,BMSCs induced14days group:the indicators, trace insulin detect West blot has obvious advantages compared to the other groups,The results suggest that BMSCs induced14days are more suitable time and status for the transplanted than the other groups.