The Role Of Stem Cells Transplantation On Induction Of Regulatory T Cells In Allogeneic Heart Transplantation

Part I Mark the rat bone marrow-derived mesenchymal stem cells with superparamagnetic iron oxide nanoparticlesObjective:To mark the rat bone marrow-derived mesenchymal stem cells with superparamagnetic iron oxide nanoparticles and observe the influence of markers on stem cells.Methods:Rat bone mesenchymal stem cells were isolated from femoral and tibial bone marrow of rats weighing200-250g with the application of adherent culture technique. Observe the subculture growth of cells by microscope. With the third generation of messenchymal stem cells taken, expression of CD14, CD29, CD31, CD34, CD45and CD90on the surface of stem cell was detected respectively by the flow cytometry (FCM). Mark stem cells by superparamagnetic iron oxide nanoparticles solution (SP1025ug/ml, PLL0.75ug/ml) and observe them by transmission electron microscope(TEM). Through differentiation induction of all labeled stem cells and that of all unlabeled stem cells into osteoblasts and adipocytes, assess the effect of iron oxide nanoparticles mark on the function of stem cells.Results:Rat mesenchymal stem cells can be successfully isolated and purified by means of adherent culture technique. All the cells are in good condition with positive expression on the surface of CD29, CD90and no expression of CD14, CD31, CD34and CD45, which is consistent with the feature of mesenchymal stem cells. The shape of nano-iron marked cells observed by TEM displayed well, with SPIO particles visible in endochylema. All labeled or unlabeled cells have good osteogenic and adipogenic differentiation.Conclusions:SPIO of25ug/ml and PLL of0.75ug/ml could well label rat mesenchymal stem cells, which has no significant influence on the expression of surface molecule and basic biological function of osteoblasts and adipocytes differentiation.Part II Experimental study of influence of rat bone marrow mesenchymal stem cells on T cell function in vitroSection I The effect of mesenchymal stem cells on proliferation of T cellsObjective:To examine the influence of rat bone marrow mesenchymal stem cells on T-lymphocytes proliferation in vitro by mixed lymphocyte reaction.Methods:Taking5×104SD rat T-lymphocytes as responding cells and5×104Wistar rat T-lymphocytes as stimulated cells, establish a one-way mixed lymphocyte reaction to which add different amount of Wistar rat bone marrow mesenchymal stem cells. Divide the above mentioned into different groups, with SD rat T-lymphocytes cultured alone in group A, SD rat T-lymphocytes and Wistar rat T-lymphocytes in group B, SD rat T-lymphocytes, Wistar rat T-lymphocytes and5×102Wistar rat MSC in group C, SD rat T-lymphocytes, Wistar rat T-lymphocytes and5×103Wistar rat MSC in group D, and SD rat T-lymphocytes, Wistar rat T-lymphocytes and5×104Wistar rat MSC in group E. After72hours of mixed culture, cell proliferation was tested by H3infiltration method.Results:after mixed lymphocyte reaction, T cell proliferation in group B, C, D, E has been significantly increased, which is statistically significant different from that in group A. Moreover, cell proliferation in groups adding mesenchymal stem cells has gradually been decreased, with significant differences between groups.Conclusions:Rat bone marrow mesenchymal stem cells can suppress T-lymphocyte proliferation in vitro in dose-dependent manner.Section II The influence of mesenchymal stem cells on subpopulations of T cellsObjective:To examine the influence of rat bone marrow mesenchymal stem cells on the subpopulations of T-lymphocytes by mixed lymphocyte culture in vitro.Methods:The same mixed lymphocyte culture system was established as that introduced in section I. After72hours of mixed culture, cell proliferation was tested by H3 infiltration method. The ratio of CD4+CD25+T cells was detected by FCM and the level of IL-2, IL-4, IL-10, TGF-β1and IFN-yin the clear liquids was examined by ELISA method in each group. After which, CD4+/CD8+T cell ratio of each group was calculated.Results:after mixed lymphocyte reaction, cell ratio of CD4+CD25+T and that of CD4+/CD8+T gradually increased, with statistical differences in group C, D and E. Level of IL-2and IFN-yhas gradually been lowered in group B, C, D and E, but that of IL-4, IL-10and TGF-β1has gradually been improved, with statistical differences between groups.Conclusions:Rat bone marrow mesenchymal stem cells can increase the number of CD4+CD25+regulatory T cells and enhance the ratio of CD4+/CD8+T cells in dose-dependent manner. It is related to the increase of TGF-(31, IL-4and IL-10, decrease of IL-2and IFN-ylevel and differentiation of Th cells into Th2.Part Ⅲ The experimental study of combined intrathymic and intravenous injection of mesenchymal stem cells to induce immune tolerance in rat heart allotransplantationObjective:To study the effect of rat bone marrow derived mesenchymal stem cell transplantation pretreatment on induced immune tolerance in rat heart allotransplantation to explore the possible molecular mechanism.Methods:Establish Wistar rat’s-and-SD rat’s allogeneic heterotopic heart transplantation model. One week before the hear transplantation, respectively take nano-iron marked rat bone marrow mesenchymal stem cells (referred as’donor’) to give the intrathymic and intravenous injection to SD rats (referred as’receptor’). The followings are experimental groupings. In group A (the control group), only perform Wistar rat’s and SD rat’s heterotopic heart transplantation. In group B (the intravenous injection group), MSCs1×106/0.2ml from the Wistar rat was injected to SD rats through caudal vein. Then, perform allogeneic hetertopic heart transplantation between the Wistar rat and the SD rat in a week. In group C (the intrathymic injection group), MSCs5×105/0.1ml from the Wistar rat was injected to the thymus of SD rats. Then, follow the same operation with that in group B. In group D (the intravenous plus intrathymic injection group), MSCs1×106/0.2ml from the Wistar rat was injected to SD rats through jugular vein. Then, follow the same operation with that in group C. Observe the survival time of cardiac allograft by means of palpation. Respectively take isolated lymphocytes of cardiac allograft from which further extract RNA on the first, third and fifth day after transplantation. By relative quantitative RT-PCR, examine the level of IL-2, IL-4, IL-10, IFN-γ, miR-7a, miR-155, miR-182, miR-183and miR-434-3p in each group. Respectively draw peripheral blood, test the ratio of CD4+CD25+Foxp3+T cells by FCM. What’s more, detect the distribution of the marked stem cells after transplantation by MRI and Prussian blue staining.Results:After rats’allogeneic heterotopic heart transplantation, the survival time of grafts in group B, C, D was significantly longer than that in the control group, which has statistical differences. The ratio of CD4+CD25+Foxp3+T cells has gradually been increased, significantly obvious on the fifth day, with statistical differences. The level of miR-7a, miR-182and miR-183on the first, third and fifth day after transplantation has gradually been improved while the level of miR-434-3p has been gradually decreased. Statistically significant differences exist at different time points, but the difference is not obvious at the same time point in each group. The growth of miR-155in group B, C and D has been declined on the third and fifth day, which has statistical differences compared to the control group, with the differences in group D more obvious. Compared to the control group, the level of IL-2and IFN-ysignificantly declined on the fifth day after transplantation, with statistical differences in group B and significant differences in group C, D. The level of IL-4in group C and D has been increased which also has statistical differences. In group D, the level of IL-10has been increased which has significant differences. The SRY gene from donor can be detected in celiac lymph nodes in group B and C. The decreasing number of marked stem cells could be observed in thymus and transplanted heart by MRI and Prussian blue staining.Conclusions:Expression of miR-155, miR-182, miR-183and miR-7of receptor increases while that of miR-434-3p decreases after rats’ allogeneic heart transplant. The number of CD4+CD25+regulatory T cells can be increased by means of both intrathymic and intravenous injection from MSCs, which can also decrease expression of miR-155in receptors, promote expression of IL-4, IL-10in allograft and lower expression of IL-2、 IFN-y and chrism formation. It is proved that the combined intrathymic and intravenous injection of mesenchymal stem cells is a more effective way to prolong the survival time of cardiac allograft.