The Research Of Transdifferentiation And Security Of Human Bone Marrow Mesenchymal Stem Cell Transplantation In Type1Diabetes Mice Modles Of Nod/Ltj Mouse

Objective:1.On the base of our previous work,we confirm further the effect of human bone marrow mesenchymal stem cells (hBMSC)transplantation in type1diabetes mice modles of NOD/Ltj mouse(Blood glucose,Serum insulin and insulin in pancreas islets);2.We investigate the underlying mechanism of hBMSC in three aspects:Immunoregulation of hBMSC:the propotion of CD4+CD25+FOXP3Treg,the propotion of CD4+/CD8+T lymphocyte,the balance state of Thl/Th2;3.To observe the homing phenomenon of hBMSC and the ability of transdifferentiation;3.To observe the security of MSC transplantation(tumorigenicity et al).Methods:Established type1diabetes models in NOD mice by injecting with40mg/kg streptozotocin(STZ) intraperitoneally for consecutive five days, three days later, blood glucose was measured,these mice whose non-fasting blood glucose>13.9mmol/l for2consecutive days were diagnosed as diabetic mice, and then were randomly divided into two groups:Diabetes Control group(n=24mice:0W=6,2W=6,4W=6,6W=6); Bone Marrow Mesenchymal stem cells Transplantation group(n=12mice:2W=64W=6BMSC2*106in100ui saline);four time periods(0W:the time to be the models、2W:the two week after transplantation or the two week after being models%4W:the four week after transplantation or the four week after being models、6W: the six week afte transplangtation or the six after being models).Mice were transplanted with BMSCs via tail vein. Blood glucose and weight were monitored every week.All the mice were killed and their spleen lymphocytes were collected in different stages.Then Collect the blood from orbital venous.The proportion of Treg cells,CD4+/CD8+Tc cells were analyzed by flow cytometry. The serum levels of cytokinesTh1/Th2were determined by ELISA.Insulin of serum was also determined by ELISA.The pancreases were dyed by HE to be observed the pancreatic structure,and IHC method was used to detect the content of insulin in the islets.Frozen sectin of pancreases and kidney were observed by fluorescence microscope to explore the homing phenomenon of hBMSC,then IF(anti-human insulin) was tested to observe whether hBMSC could secrete human insulin.Results:1.Effect of transplantation(We will state that in three aspects:Blood glucose,serum insulin and insulin in pancreas islets):(1)Blood glucose:Three days after the first STZ-injection, the blood glucose level gradually increased. The blood glucose levels of the transplantation groups were lower than that in control group. The difference was significant between transplantation groups and control group in1-4weeks.The results of IPVTT showed that blood glucose on0,60,120min in2w and that on0,120min in4w in transplantation group were lower than that in control group.AUCG reduced in transplantation group(P<0.05).In addition,the weight of mice in transplantion group in3w was higher than that in control group(.P<0.05).(2)Serum insulin:More iusulin was detected in transplanted group by ELISA,but had no statistical differences.(3)Insulin in pancreas islets:After mading models success,the pancreas structure was much damaged. As diabetic medical history extend,islet structure was damaged more seriously.Islet number reduced obviously. But after transplantation, Islet number was more increased than that in control group.The islet structure was relatively complete.and a few new islets structure were found.More iusulin was detected in transplanted group by Image-ProPlus,but had no statistical differences.2.The observation of inmunoregulation(in aspects of CD4+/CD8+Tc,Treg, Th1/Th2):(1)CD4+/CD8+Tc:The ratio of CD4+Tc cells was significantly reduced in the transplantation groups in different stages(P<0.05)and CD8+Tc cells had significant difference in4 weeks(P<0.05).the control group.(2)Treg cells:The ratio of Treg cells in spleen all increased in transplantation groups in different stages(P<0.05).(3)Thl/Th2:Compared with the control group, the levels of IFN-yand TNF-a were lower, and the level of IL-5, IL-6,IL-10were higher than those in the experimental group.3.Homing phenomenon and transdifferentiation ability:Frozen sectin of pancreases and kidney were observed by fluorescence microscope,some hBMSC marked by CFSE could be observed in pancreases and little seen in kidney.Then IF(anti-human insulin) were tested and no human insulin was observed in hBMSC in pancreases.4.Observation of security:We found tumors in knee of two mice in transplantation and the results of HE show that the bases of tumors are osteoclast like cell and chondrocyte-like cells.Conclusions:1.Transplantation BMSCs can reduce hyperglycemia in type1diabetes model mice; Insulin in serum and pancreas islets are more increased than those in control group.2.After BMSCs transplantation, the ratio of Treg cells increased and CD4+、CD8+Tc cells reduced compared with those of control group;The levels of IFN-yand TNF-a increased and the levels of IL-4,IL-5,IL-10increased, which might be the immune mechanism of BMSCT therapy in TID.3.hBMSC might homing to damaged pancreas islets but not to trandifferent into insulin-secreting cell.4.MSC transplantation might have tumorigenicity, so we must concentrate on alerting the further security of MSC transplantation in clinical work.