The Immunoregulation Effects Of Bone Marrow Mesenchymal Stem Cells On The Lymphocytes Of Rats With Type 1 Diabetes Mellitus In Vitro And In Vivo

Diabetes mellitus is a heterogeneous primary disorder of carbohydrate metabolism with multiple etiologic factors that generally involve absolute or relative insulin deficiency or both. It includes type 1 diabetes mellitus and type 2 diabetes mellitus. Type 1 diabetes mellitus is a multifactorial chronic disease, which results from the selective autoimmune destruction of insulin-producingβ-cells. The gold standard treatment for type 1 diabetes mellitus is intensive insulin replacement; however, serious complications can occur even under the most careful supervision. Clinical islet transplantation performed under the Edmonton protocol, a steroid-free immunosuppressive regimen first described by Shapiro et al. in 2000, represented a groundbreaking innovation in the field of islet transplantation to cure diabetes. However, the curative solutions are complicated by the undesired side effects of life-long immunosuppression, organ shortages and in the case of islet transplantation, the shortage of human islet donors and difficulties in islet cells preparation.Mesencymal stem cells are pluripotent stromal cells that have the potential to give rise to cells of diverse lineages. They have also been noted to possess the ability to impart profound immunomodulatory effects in vivo.The recent researchs indicate that the Immunoregulation effects of mesenchymal stem cells are closely related to the number and activity of regulatory T cells. On the other hand, recent studies indicate that defects in both number and activity of regulatory T cells are causally related to the development of T1DM. Thus,increasing the number of Tregs by adoptive transfer can be used to prevent and treat T1DM. Because of their developmental plasticity and immunomodulatory functions, the notion of MSCs-based therapeutic intervention has become an emerging strategy for immune-mediated disorders and, in particular, type 1 diabetes mellitus.Objective: Observe the effects of bone marrow mesenchymal stem cells on the lymphocytes of rats with type 1 diabetes mellitus both in vitro and in vivo and the impacts of MSCs and IPCs co-transplantation in cell therapy of type 1 diabetes mellitus.Isolation,cultivation and identification of rat MSCs in vitro: MSCs were isolated from SD rat, cultured in vitro, purified and then identified by observing the phenotypes with flow cytometry(FCM). Results:The phenotypes of MSCs from normal SD rats were CD29~+,CD90~+,CD106~+,CD34~-,CD45~-, which had potential capability to be differentiation into fat cells in vitro.Conclusion: MSCs can be cultured stably,expanded quickly and have high purity with the characteristic of their adhesive process onto the culture dish. The Immunoregulation effects of bone marrow mesenchymal stem cells on the lymphocytes of rats with type 1 diabetes mellitus in vitro: The third passage MSCs were planted in 24-well plates. After treated with Mitomycin C. MSCs were co-cultured for 72h with the T1DM rat's lymphocytes activated by phytohemagglutinin (PHA). There were five groups, comprising lymphocytes/ MSCs:1:1, 10:1, 100:1 groups, lymphocytes+PHA group(proliferation group), and lymphocytes group(blank group).The proliferation of lymphocyte was measured by MTT method.FCM analysis were applied to investigate the apoptosis,cell cycle and the proportion of CD4~+CD25~+ regulatory T cells of the T1DM rat's lymphocytes after co-cultivation. Results: MSCs obviously inhibited the lymphocyte proliferation stimulated by PHA. The inhibitory effect was the strongest at 1:1 of lymphocytes and MSCs(C group). In the co-culture system, most of the lymphocytes were arrested at G0/G1 phase.The apoptosis of lymphocytes(58.05±0.89)% in C group increased significantly than that of the control group (43.35±0.86)%(P<0.05)as well as the proportion of CD4~+CD25~+ Tregs[(22.76±1.15)% VS(5.80±0.68)% ]. Conclusion: In vitro, MSCs can obviously inhibit the T1DM rat's lymphocytes proliferation stimulated with PHA, via increasing the proportion of CD4~+CD25~+ Tregs.The impacts of MSCs and IPCs co-transplantation in cell therapy on type 1 diabetes mellitus rats: The third passage MSCs were cultivated in vitro mimicking microenvironment(Rat Pancreatic Extract was added), in order to obtain greater number and better functioning IPCs. We transplanted IPCs(2×10~6 cells/rat via tail vein) alone or in combination with MSCs(3×10~6 cells/rat,via tail vein) into the STZ-induced T1DM rats. The levels of serum IL-6, IL-10, INF-γ,and TGF-β1, and the CD4~+CD25~+FOXP3~+Tregs Percentage were examined by ELISA and FCM. Results: The IPCs obtained from RPE added group had greater number and better function. Co-tansplantation the MSCs and IPCs had more effective in cell therapy of diabetes. The IPCs could rapidly reverse hyperglycemia in diabetic rats and the MSCs could alter T cell cytokine pattern and up-regulated the level of CD4~+CD25~+FOXP3~+Tregs percentage, then exert unique immunosuppressive effects. Conclusions: The microenvironment in which the RPE was added can promote the differentiation of MSCs into better IPCs in vitro. The IPCs can be a potential source for the shortage欧of donor islets and the MSCs possess the ability to impart profound immunomodulatory effects in vivo,which protect the graft and the newly formed islets.In summary, MSCs can be cultured stably,expanded quickly and have high purity with the characteristic of their adhesive process onto the culture dish. Our study presents the phenomenon that MSCs can be induced to transdifferentiate into IPCs both phenotypically and functionally by microenviromental manipulation. We have also shown that transplantation with neither IPCs nor MSCs alone is sufficient for treatment of diabetes, co-tansplantation the MSCs and IPCs have more effective in cell therapy of diabetes. Obviously, there are still a number of fundamental knowledges about MSCs need to be understood before they can be safely and effectively applied in clinical setting, but because of their developmental plasticity and immunomodulatory functions, the notion of MSCs-based therapeutic intervention has become an emerging strategy for immune-mediated disorders and, in particular, type 1 diabetes mellitus.