The Protection Of Islet Grafts By Bone Marrow Mesenchymal Stem Cell

Transplantation of islets of Langerhans has represented a viable therapeutic approach for the treatment of diabetes since 2000. Different from other organ transplantation,the aim of islet transplantation is to improve the quality of the life of recipients. It would not be beneficial for patients with diabetes mellitus if severe side effect occurred due to the management of immunosuppressive agent. So immune tolerance seems to be better option.Bone marrow mesenchymal stem cells have been discovered multipotential in immunoregulation. They can suppress the antigen-specific or nonspecific lymphocyte proliferation through cell contact with other immune cells or by secreting kinds of soluble molecules. They are able to inhibit the differentiation and maturation of antigen presenting cells and inducing antigen specific Treg cells’ formation, therefore modulate the immune response towards an anti-inflammatory direction. Because of the powerful immunoregulation ability, BM-MSCs present great potential in clinical application in transplantation and replacement therapy. It was reported that BM-MSCs co-transplanted with islets could improve the survival time of islet grafts through modulating immune tolerance of islets grafts. The mechanism may be related to cell contact or cytokines secretion. However due to the complicated function of BM-MSCs,there was not a conclusion. It was also reported that being regarded as a new CD4+ T cell subset with independent source of differentiation, the T follicular helper cells(Tfh)had been confirmed to play an important role in multiple autoimmune diseases,transplantation immune tolerance and tumor immunity. Nevertheless whether the Tfh cells was related with Type I diabetes mellitus(T1DM) was unclear. Therefore we were considering whether Tfh cells were involved in the development of Type I diabetes?And whether the protection of islet grafts function by BM-MSCs was because theymodulated Tfh cells differentiation and further regulated the immune homeostasis? So we conducted experiments on this issue. First of all, we improved the islet isolating method on the basis of Gotoh islet isolation technology. The new islet isolation process is much simple and efficacious and the experiment success rate is improved. At the same time we adopted the whole bone marrow culture method to separate BM-MSCs and identified through fluorescence activated cell sorting(FACS). On the basis of the experiments above, we established the islet transplantation model of Non-obese Diabetic mice(NOD), researched the results of different BM-MSCs/islet co-transplantation methods and performed a preliminary study of BM-MSCs modulating Tfh cells differentiation. The purpose of our study was to illustrate that the mechanism of BM-MSCs protecting the islet grafts was regulating the Tfh cells and provide new thoughts for treatment of Type I diabetes.Part I Mouse islet isolation and purificationObjective: To establish a better method of isolation and purification of C57BL/6mouse islets.Methods: The 24 G cannulas was inserted at the duodenal part of pancreatic duct.After collagenas IV perfusion,digestion and washing, histopaque-1077 and Hanks solution were prepared for gradient centrifugation. The islet was suspended in the interface of the two solution.The purity of islets was accessed by dithizone(DTZ)staining and viability by acridine orange(AO) and propidium iodide(PI)fluorescence staining. The function of islet was determined by glucose stimulated test.Results: The total number of islets from one donor was 195.00±14.95 IEQ/mouse(purity>90.00%). Islet isolated was stained red by dithizone. AO/PI staining showed that the alive cells were more than 95.00%. The secretion amount of insulin in the high glucose(23.00±3.90 ng.ml-110islet-1.45min-1) stimulation was two folds more than lowglucose stimulation(10.10±1.40 ng.ml-110islet-1.45min-1).Conclusion: Insertion of the 24 G cannulas at the duodenal part of pancreatic duct was much better than insertion at the bile duct. Histopaque-1077 and Hanks solution was an easier gradient centrifugation for operation and the islets isolated were functional.Part II Mouse bone marrow mesenchymal stem cells isolation,culture and identificationObjective: To introduce an optimal and efficacious method for BM-MSCs primary culture in vitroMethods: Mouse bilateral femur and tibia were separated. Bone marrow was extracted from the marrow cavity and cultured in culture dish. Cell morphology and quantity were observed daily. BM-MSCs were identified by FACS with specific cell markers CD29、CD117、CD44、CD31 and Sca-1.Results: The total amount of BM-MSCs from one donor was(5.35±0.40)×105.Cell grew in good morphology and quantity. BM-MSCs expressed CD29(96.04%) 、CD44(96.21%) and Sca-1(95.72%) except for CD117(0.13%) and CD31(0.23%).Conclusion: The whole bone marrow culture method was easily operated and achieved BM-MSCs with normal morphous. This achievement permit further experiments on exploring immunologic characteristics of BM-MSCs and the co-transplantation with islets and BM-MSCs could be carried out.Part III The effect and mechanisms of BM-MSCs on protection of allograft isletsObjective: To investigate the effect and mechanisms of BM-MSCson protection of allograft islets in different transplantation plans.Methods: Five Groups were set: Group A: control Group of normal NOD mice;Group B: the diabetic Group; Group C: islets alone Group;Group D: BM-MSCs/islets co-transplanted Group; Group E: BM-MSCs i.v. administration and BM-MSCs/islets co-transplanted Group. Islets or islets and BM-MSCs were transplanted under recipients’ kidney capsule respectively. The blood glucose of recipients were monitored. Glucose tolerance test was performed at 7th day after surgery.Mouse spleen and peripheral blood were obtained on the 7th day after surgery.ELISA was used to test the level of diabetes autoantibody GAD65 Ab and IAA. Tfh cell count was tested by FACS.ICOS and Bcl-6 were tested by Western blot. The grafts were obtained for HE staining Immunohistochemistry and Immunofluorescence staining.Results: Group D(23.67 ± 2.77 days) and Group E(34.00 ± 4.51 days)exhibited significant longer survival time of grafts than Group B(11.67±2.27 days). Recipients of Gruop C 、 Group D and Group E exhibited a function loss of grafts at 14.0th 、 26.0th and 42.0th day respectively.Apparent hypoglycemia was observed in Group C(3.5± 1.3 mmol/L) on the second day after surgery,but both Group D(6.5± 1.4 mmol/L) and Group E(7.1± 1.4 mmol/L) stayed normoglycemia(5.0~20.0 mmol/L). The Glucose tolerance test showed that all transplantation Group could control the blood glucose,but Group D(18.4 ± 0.9 mmol/L) and Group E(18.0 ± 0.7mmol/L) controlled the blood glucose more steady than Group C(21.8±1.3mmol/L). The difference between Group D and Group E was not significant.In comparison with Group C,the diabetic autoantibody level was much lower after BM-MSCs co-transplanted in both co-transplant Group(GAD65:A: 0.40 ±0.02;B: 1.49 ± 0.03;C: 1.20 ± 0.09;D: 0.82 ± 0.03;E: 0.42 ± 0.03 OD 450 nm and IAA:A: 0.31 ± 0.03;B: 1.31 ± 0.06;C: 1.03 ± 0.07;D: 0.70 ± 0.04;E: 0.32 ± 0.03 OD450nm).The Tfh cells level were much lower in Group E(A:4.35±1.54%;B:24.55±5.41%; C:23.87±6.67%; D:9.67±1.34%; E:4.27±0.59%). The expression ofICOS and Bcl-6 decreased in Group D and Group E than Group B and Group C. All staining showed that the grafts in Group E exhibited a more normal morphous, higher insulin secretion and less lymphocytes infiltration than Group C and Group D.Conclusion: MSC may protect the islet transplants and induce the islet grafts immune tolerance by modulating the Tfh cells and down-regulating the autoantibody level. The BM-MSCs i.v. administration and BM-MSCs/islets co-transplanted plan was more effective than both islet alone transplantation plan and BM-MSCs/islets co-transplanted plan.