Syngenic Islet-transplantation In NOD Mice And Derivation Of Bone Marrow Mesenchymal Stem Cells From NOD Mice

Diabetes is becoming a serious threat to human being’s health.IDF Diabetes Atlas 2015 showed that about 4.15 billion adults worldwide suffer from diabetes,and forecasts worldwide diabetes patients will reach 6.42 billion in 2040.At present,the total number of diabetes patients in China has reached 1.096 billion,ranking first in the world.Diabetes mellitus is generally divided into two categories:type 1,called insulin-dependent diabetes,and type 2,called non-insulin-dependent diabetes.Patients with type 1 diabetes require lifelong insulin therapy because of the autoimmune destruction of insulin-producingβ-cells.Although hyperglycemia can be improved by insulin treatment,exogenous insulin injection cannot precisely mimic the insulin secretion of endogenous β-cells.Intensive insulin therapy can prevent or slow the progression of long-term diabetes complications,but meanwhile it significantly increases the risk of severe hypoglycemia.Although pancreatic islet transplantation can re-establish the endogenous insulin secretion in type 1 diabetic patients,the shortage of pancreas donors remains a major challenge in clinical islet transplantation.The aim of this study was to investigate the changes in blood glucose levels after syngenic islet-transplantation in NOD mice and the derivation of MSC from NOD mice.Part Ⅰ Establishment and Functional Detection of Isolation and Purification of NOD MiceObjective:This investigation was initiated to establish a stable and efficient method for isolation and purification of islets in NOD mice.Methods:The pancreatic islets were isolated from the pancreas by common bile duct intraductal collagenase P digestion and purified by discontinuous ficoll PM 400 density gradient centrifugation.Islet yield and purity were determined by dithizon(DTZ)stain.The endocrine secretory function was assessed by insulin secretion in either low or high glucose stimulation.Results:After isolation and purification,average isolated number was(116 ± 12)islets/pancreas.The average purity of islets were 90%.Insulin secretion from NOD mouse islets was(0.489 ± 0.0295)ng/islet in high glucose incubation,which was significantly higher than that of low glucose environment[0.365 ±0.0577 ng/islet,P<0.01)].The average insulin stimulation index was 1.33.Insulin secretion from KM mouse islets was(1.052 ± 0.033)ng/islet in high glucose incubation,which was significantly higher than that of low glucose environment[0.391 ± 0.0125 ng/islet,P<0.01)].The average insulin stimulation index was 2.69.Conclusions:Our study provides a method of the isolation and purification of NOD mice islets,which can obtain high-purity,high-yield,and high-viability islets.Meanwhile,our results indicated that islets derived from normal NOD mouse have a damaged insulin secretion function.Part Ⅱ Syngenic Islet-transplantation in NOD MiceObjective:This investigation was initiated to develop a method for syngenic islet-transplantation in NOD mice.Methods:14-week old female diabetic NOD mice served as islet graft recipients.Diabetes was defined as fasting blood glucose>16.8 mmol/L on 2 consecutive days.Islet donors were male NOD mice,7-8 weeks of age.The female NOD mice were then randomly allocated into two groups,the PBS-injected sham group(sham-operated group,n=6)and islet transplant group(transplant group,n=6).A total of 300 islets per mouse were implanted under the renal capsule.Then,Blood glucose,body weight changes,and glucose tolerance test was performed two weeks after transplantation.Mice were sacrificed at 70 days after transplantation.HE staining and immunofluorescence staining were performed in paraffin-embedded graft biopsy sections to observe the survival of transplanted islets.Results:In the control group,blood glucose was maintained at the level of 25mmol/l.In transplant group,66%of mice can maintain blood glucose at the level of 15 mmol/1 for two weeks,and 33%of mice can maintain blood glucose at the level of 10 mmol/l for 7 weeks.Furthermore,immunostaining assay revealed that insulin positive cells could be observed on graft biopsy sections.Conclusions:The method for syngenic islet transplantation in NOD mice was successfully established.Part Ⅲ Isolation and culture of bone marrow mesenchymal stem cells from NOD mouseObjective:This investigation was initiated to develop a method for isolation and culture of mesenchymal stem cells from NOD mouse bone marrow.Methods:We isolated BMSCs by using adherent selecting from NOD mouse bone marrow and bone fragments.Then,the phenotypes of MSCs were identified by flow cytometry,adipogenic differentiation and osteogenic differentiation in vitro.Results:Our results showed that MSCs could be successfully derived from NOD mouse bone marrow and bone fragments.To further characterize the phenotype of cultured the above MSCs,we examined the surface markers by flow cytometry and the differentiation potentials.Although our results indicated that there was a lower expression of CD 44(27.26%± 6.00%)and CD 29(17.23%± 5.19%)in MSCs from bone marrow,and CD 44(30.33%± 6.63%)and CD 29(29.00%±12.49%)in MSCs from bone fragments,these cells could be readily differentiated into osteocyte and adipocyte cells by culturing in appropriate induction media.Conclusions:Although NOD mous MSCs derived from bone marrow or bone fragments did not express typical mouse MSC surface markers,they could be successfully differentiated into osteocyte and adipocyte cells.