Experimental Study Of Rapamycin On The Differentiation And Proliferation Of CD4~+CD25~+ Regulatory T Cells In Mice

Allograft rejection has been the largest problems since the birth of the transplant medicine.Each improvement of anti-rejection push transplant medicine forward.At present,efficient immunosuppressants can control acute rejection effectively,But have poor reaction on the hyperacute rejection and chronic rejection,and the lifelong use of immunosuppressant drugs can cause adverse reactions,such as the bone marrow suppression,cancer,infection,chronic loss of the transplanted organ function, as well as their own drug side effects and high costs.Since Medawar made the concept of transplantation immune tolerance,which immune system non-response specificity to allogenic transplantation antigen and maintain normal defense function to the other antigen or pathogen under no immunosuppressants,it has been the ultimate goal of the pursuit of organ transplantation.Main mechanism of body maintain theirself immune tolerance includ central and peripheral tolerance,the former mainly is cell clonal deletion occurred in the central immune organ;the latter including clonal deletion,clonal anergy,immune ignorance,immune suppression.Immunosuppression play a role of suppression of the immune system by negative adjustment of regulatory T cells.Treg cells is a kind of immune regulatory function T-cell subsets differentiating type of Th1 and Th2,having function of Immune incompetence and immune suppression.IT Mainly play immunosuppressive effects through two mechanisms,one is cell contact:TGF-βof membrane surface binding receptor of TGF-βof the target cell surface after Treg cell activation,suppressing proliferation and activation of effector T-cell and mature of dendritic cells;meantime,its surface expression CTLA-4 can also play a suppressing function by binding the CD80 and CD86 the another is secretion Cytokine:Treg cell suppress antigen-presenting of APC and activation of the effector T cells by release cytokine,such as IL-10,TGF-βand so on.Treg cells come from the differentiation of T cells in thymus,also can come from differentiation of periph resting T cells.Transcription factor Foxp3 gene participate the central and peripheral formation process of Treg cells,and impact function of Treg cells.Rejection or tolerance is decided by the balance of effctor T cells and Treg cells. For a long time,immunosuppressive treatment main purpose is to combat the immune system of receptor,suppressant,such as CsA,etc.can suppress rejection, but may also inhibit the generation of Treg cells,which is considered closely related to long-term graft acceptance,may block spontaneous formation of body immune tolerance after transplantation.Animal experiments show that,in the absence of immunosuppression circumstances,graft can be accepted by receptor,and these models in the application of some of high-dose immunosuppressant,immune tolerance of receptor on graft was blocked.Search inhibiting rejection,while not inhibiting Treg cells generated,does not preventing formation of graft immune tolerance will be the new research direction.This study analyzed clinical common immunosuppressant cyclosporine A(CsA) and Rapa affect the proliferation of CD4~+ CD25~+ T cells and expression of Treg cell transcription factor Foxp3 in vivo and in vitro,and affect the immune tolerance,clinical use as a guide to improve organ transplant survival period and provide a basis for ideas.PARTⅠ.Experimental study of rapamycin on the differentiation and proliferation of CD4~+CD25~+ regulatory T cells in mice in vitroObjective:To explore the feasibility and possible mechanism of induction of immune tolerance with rapamycin by investigating effect of CD4~+CD25~+Treg cells value and the expression of Foxp3 trader rapamycin or cyclosporin A co-cultured with CD4~+ T cells.Methods:Take spleen from C57BL/6 mice under sterile conditions,isolated mononuclear cells,negative separate CD4~+ T cells by immunomagnetic beads,divide into control group,rapamycin group and cyclosporine groups co-culture with anti-CD3,anti-CD28 and IL-2 for 7d.detect CD4~+CD25~+ Treg cells value on flow cytometry and expression level of Foxp3mRNA by semi-quantitative RT-PCR.Results:Compared with the control group,the proportion of CD4~+CD25~+ Treg cells in the CD4~+ T cells of cyclosporine group significantly decreased(3.72%vs7.42%,p＜0.01);the proportion of CD4~+CD25~+ Treg cells in CD4~+ T cells of rapamycin group significantly increased (11.47%vs 7.42%,p＜0.01);expression of Foxp3 mRNA of rapamycin group was significantly higher than that of cyclosporine group(P＜0.01) was significantly higher than that of the control group(P＜0.01);expression of Foxp3 mRNA of cyclosporine group compared with the control group significantly lower(P＜0.05). Conclusion:rapamycin promote the proliferation and growth of CD4~+CD25~+ Treg cells in vitro,while cyclosporine A inhibit the proliferation and growth of CD4~+CD25~+ Treg cells.Foxp3 positively correlated with CD4~+CD25~+ Treg cells in vitro.PARTⅡ.Experimental study on the effect of rapamycin on regulatory CD4~+CD25~+T cells in mice heart transplantation modelObjective:To explore the feasibility and possible mechanism of induction of immune tolerance with rapamycin by investigating effect of survival time of heart CD4~+CD25~+Treg cells value and the expression of Foxp3 under rapamycin or cyclosporin A to heterotopic heart transplantation model in mice.Methods: Establish male BALB/C→C57BL/10 mice neck heterotopic heart transplantation model by Cuff method,in accordance with postoperative medication,receptor divided into 3 groups(n=10):control group(no treatment after),rapamycin treatment group,Cyclosporin A treatment group.Records of survival time of graft heart,Take spleen when graft heart don't beat or 14 d after operation,isolated mononuclear cells,detect CD4~+CD25~+ Treg cells value on flow cytometry and expression level of Foxp3mRNA by semi-quantitative RT-PCR.Results: Compared with the control group,rapamycin and cyclosporine group were significantly improved survival time of cardiac allograft(p＜0.01),while no significant difference between the two(p＞0.05),Compared with the control group, the proportion of CD4~+CD25~+ Treg cells in the CD4~+ T cells of cyclosporine group significantly decreased(3.80%vs7.24%,p＜0.01);the proportion of CD4~+CD25~+ Treg cells in CD4~+ T cells of rapamycin group significantly increased(12.07%vs 7.24%,p＜0.01);expression of Foxp3 mRNA of rapamycin group was significantly higher than that of cyclosporine group(P＜0.01) and significantly higher than that of the control group(P＜0.01);expression of Foxp3 mRNA of cyclosporine group compared with the control group significantly lower(P＜0.01).Conclusion: rapamycin or cyclosporin A both were significantly improved survival time of cardiac allograft after heart transplantation in mice,there are no obvious gap in survival time between two.rapamycin promote the proliferation and growth of CD4~+CD25~+ Treg cells in vivo,while cyclosporine A inhibit the proliferation and growth of CD4~+CD25~+ Treg cells.Foxp3 positively correlated with CD4~+CD25~+ Treg cells in vivo.