The Research Of Simultaneaus Islet-Mesenchymal Stem Cells Transplantation In Type Ⅰ Diabetes Mellitus Rats

PartⅠ:Vitro ExperimentExperiment One:Isolation and cultivation and identification of the Islets of ratsObjectives To study the method for isolation and purification and the evaluation of islets quality in vitro.Methods 1.Rats' islets were isolated by the solution of the collg chase injected through the common bile duct and purified by Ficoll's discontinuous density gradient centrifugation.2.The identification of islets were studied with dithizone(DTZ) and with acridine orange(AO)-propidium.Iodide(PI).Results The islets of SD rat were round,like island and blood-red with staining DTZ.Diameter was 50-200 um.The range of the number of islets was 300-500 islets/rat. Islets purity was over 95%.Conclusions DTZ and AO-PI combined staining may exactly identify the purity,amount and viability of islets.Isolation by the collagenase and purification by Ficoll's discontinuous density gradient centrifugation can produce a high yield of viable purified islets in rats.Experiment Two:Isolation and cultivation and identification of the BM-MSCs of ratsObjective To explore a new method for the isolation and purification in vitro of mese nchymal stem cells(MSCs) from rat bone marrow and analyse their properties.Method BM-MSCs were isolated and purified from the bone marrow of SD rats by density gradient centrifugation and by adhering to the culture plastia After successive subculture and amplification,the morphology was observed under phase contrast micr oscope,then induce them to differentiate to osteoblast and adipose cell.Result The isolated BM-MSCs were mononuclear cells in marrow.Their living behavior was quite stable in L-DMEM containing 100 ml / L newborn bovine serum,and could be induce to osteoblast and adipose cell.Conclusion A method that of density gradient centrifugation and adhering to the culture plastia for the isolation and purification in vitro of MSCs from rat bone marrow has been established.The method can produce a high yield of viable purified BM-MSCs in rats.Experiment Three:The research of co-culture of BM-MSCs and islets of rats in vitroObjective To explore weather BM-MSCs could promote islets viability in vitro.Method The BM-MSCs and islets of SD rats were cultured in vitro divided into GroupⅠ(30IEQ+2×10~4BM-MSCs) andⅡ(30IEQ).Islets viability and insulin release after glucose challenge were tested to evaluate their functions.Result At the day3,7,14,islets viability was significantly higher in mix-cultured group than that in islets cultured alone group(P<0.05).The response to high glucose was reduced along with the culture time.Stimulating index dropped from 3.1-3.3(1day) to 1-1.6(7 day).Conclusion BM-MSCs can promote islets viability in vitro when they were co-cultureed.Experiment Four:Establishment of a streptozotocin-induced rat model of T1DMObjective To establish a streptozotocin-induced rat model of T1DM and evaluate the validity.Method 16 SD rats were randomly divided into two groups.The rats in experiment group were intraperitoneally injected with streptozotocin(STZ)(60mg/kg).The control group were treat with placebo.After three days,measure the glucose level of blood in the rats.To select rats as the T1DM model when the glucose level was more than 16.7mmol/L and continuously for more than 5 days after two continually randomly measure.Result It showed a significant increase on blood glucose,24-hour urine,and decrease on weight in experiment group.Conclusion The STZ-induced rat model of diabetes developed prolonged high blood glucose and this animal model could be used to study human T1DM.PartⅡ:In Vivo ExperimentExperiment One:Transplantation of rat BM-MSCs combined with islets to TypeⅠDiabetes Meilitus(T1DM)Objective Transplantation of rat BM-MSCs combined with islets by portal vein to treat rat model of T1DM and observation the effect.Method 32 STZ-induced SD rats model of diabetes were randomly divided into four groups:Ⅰ(1000IEQ/rat+1×10~6 BM-MSCs/rat),Ⅱ(500IEQ/rat+1×10~6 BM-MSCs/rat),Ⅲ(1000IEQ/rat) andⅣ(1×106 BM-MSCs/rat). Glucose changes of STZ-induced diabetic rats were monitored after portal vein transplantation of BM-MSCs combined with islets.Result The success rate of the operation was 100%.None rat dead in 24h.The blood glucose levels were below 16.7 mmol/L within 2-3 days after transplantation and gradually increased thereafter and the time of blood glucose levels below 16.7mmol/L were extended to(21.0±1.7 d), (15.2±1.3d),(10.2±2.0 d) in groupⅠ,Ⅱ,Ⅲrespectively(P<0.05).The blood glucose levels were not decreased after BM-MSCs transplantation alone.Conclusion It was safety and significantly decreasing the blood glucose level of the transplantation of rat BM-MSCs combined with islets by portal vein to treat rat model of T1DM.