Establishing Type2Diabetes Mellitus Rhesus Monkeys Model And Bone Marrow Mesenchymal Stem Cells Transplantation To Treat Them

Objective:1.Culturing and identifying bone marrow mesenchymal stem cells of rhesus monkeys in intro;2. Investigating the effect of SPIO and DAPI labeling and tracking on bone marrow mesenchymal stem cells (BMSCs);3.Establishing the model of type2diabetes mellitus of rhesus monkeys;4.Observing distribution of SPIO and DAPI labeled BMSCs in type2diabetes mellitus rhesus monkeys after transplantation, and find out differentiation of BMSCs distribution by infused in transplanted pancreas artery. We hope to find a kind of easy and feasible techniques and methods for future clinical BMSCs transplant therapy in diabetes.Methods:1.Culturing and identifying BMSCs:BMSCs were derived from bone marrow aspirates of healthy rhesus monkeys. Mononuclear cells were isolated using density gradient centrifugation,cultured and expanded based on plastic adherence. Cellular growth characteristics were observed and recorded under inverted phase contrast microscope. To identify most cells were BMSCs, detection and analysis of surface antigen of CD29、CD31、CD34、CD44、CD90and CD105, were performed by flow cytometry.2.Labeling and tracking on BMSCs:the culture media contained with SPIO paticles and DAPI were added to the BMSCs,the ultimate concentration of SPIO was25mg/ml and of DAPI was1ug/ml respectively. Then we observe the labeling efficiency of SPIO by prussian blue staining,intracytoplasmic iron particles was shown by the transmission electron microscope and the labeling efficiency of DAPI by fluorescence microscope.The growth ability,proliferation and viability was test by MTT.3.Establishing the model of rhesus monkeys type2diabetes mellitus: totally12healthy rhesus monkeys were randomly assigned into two groups; normal control group(n=3):the rhesus monkeys without any treatment;model group(n=9):the rhesus monkeys induced by high-glucose and high-fat diet combined with low-dose streptozotocin. Then the T2DM model group was assigned into two group:the model control group(n=3):infused2ml physiological saline;the therapy group (n=6):three model monkeys infused labeled BMSCs and three model monkeys infused unlabeled BMSCs.4.Evaluating therapeutic effect of BMSCs transplantation:transplanting the SPIO and DAPI labeled BMSCs and unlabeled MSCs into type2diabetes mellitus rhesus monkeys through pancreas artery respectively.Then we observed the location, distribution and migration about BMSCs in rhesus monkeys in vivo by magnetic resonance at the3、7、1、21days respectively.We evaluated the efficience of transplantation by determination of fasting blood glucose(FBG)、fasting blood triglyceride(TG) and total cholesterol(TC) and blood insulin、C-peptide.4weeks after infusion, each group a monkey was sacrificed randomly from the model group,the control group and the model control group, then the histological specimens of the pancreas, kidney and liver were taken.HE staining, PAS staining and masson staining were taken to investigate the morphology of the three groups monkeys.Prussian blue staining and fluorescence staining were performed with the tissue slices to find out SPIO and DAPI labeled BMSCs and observe its distribution in various organs.Results:1.Morphology observation and identification of BMSCs:under inverted phase contrast microscope,the primary cells from bone marrow were adherenced and forming colonies after3～4days.Continuing cultured for10～14days,the adherenced cells reached80%of cell fusion and showed spindle shapes and vortex condition growth.Flow cytometry detection of the adherenced passage3cells indicated high expression CD2、CD44、CD90and CD105;low expression CD31and CD34, which demonstrated the cells we got were BMSCs.2.Identification of labeled cells:labeled BMSCs grow normally,the labeled efficiency reached to95%～98%by prussian blue staining、transmission electron microscope and fluorescence microscope.3.Blood biochemical parameters of model monkeys:before modeling to12weeks after modeling,the FBG lever ranging from (3.6±0.8mmol/l) increased to (21.5±4.1) mmol/L;Blood insulin and C-peptide lever significantly reduction;TG and TC significantly increased, the changes showed significantly statistiscal difference(p<0.05).In monkeys,the serum glucose and C-peptide as well as the area under the curve(AUC) for intravenous glucose tolerance tests(IVGTT) and acute C-peptide response(ACR) were significantly statistiscal difference(p<0.05).4. Histopathological changes in model monkeys:the pathological sections of the model group pancreas showed that the morphology of islets were atrophy,the quantity of islets were reduced,the structure of islets were broken and there was many glycogen deposition in the pancreas. The pathological sections of the model group kidney showed that the lumen of glomerular capillary collapsed,obliteration mesangial region widened,and mesenterium base material mult.In addition,the volume of the the glomerulum increased and cell population mult. The pathological sections of the model group liver showed that the structure of hepatic lobule was destroyed,hepatic cord was in disorder,hepatic sinusoid was narrowed,hepatic cells exhibited fatty degeneration,some of which showed interstitial fiber fibroplasias and there was many glycogen deposition in the liver.5.Observation after cells transplantation:the labeled cells could be identified on T2W1sequence after infusion into diabetic monkeys by using3.0T MR.BMSCs were observable and could be seen for at least14days.4weeks after infusion labeled cells, the monkeys pathological sections showed that the survival of labeled cells could be easily detected in the pancreas and kidney. we can found that the cytoplasm of labeled cells were in blue color by prussian blue staining the slice and the blue bright fluorescence could be found in DAPI labeled cells by fluorescence staining the slice under fluorescence microscope.6.Blood biochemical parameters changed after transplantation:6weeks after infused cells,blood FBG、TG and TC significant decreased;insulin and C-peptide lever increased,they had statistically significant refer to control group(p<0.05).In monkeys,at the6weeks after transplantation,serum glucose and C-peptide as well as the AUC for IVGTT and ACR were significantly statistiscal difference(p<0.05) refer to model control group.Conclusion:1.Successfully cultured the high purity BMSCs(95.1%) and successfully labeled BMSCs by SPIO and DAPI.labeled efficiency was95%～98%.2.We got high achievement ratio about T2DM rhesus monkeys model made by high-glucose and high-fat diet combined with low-dose streptozotocin,in additional,the blood glucose continuously stable.3.Transplanted BMSCs could survive, migrate,differentiate and incorporate in the pancreas and kidney. MR technique is an ideal method for tracking labeled cells after infusion in vivo.BMSCs transplantation for the treatment of type2diabetes is safety and effective.