| Allograft rejection has been one of major problems for allograft survival. It is very important to actively prevent the transplant recipient from rejection and complications for their survival time and the quality of life. Organ transplant rejection includes the host anti-graft reaction (HVGR), which is commonly referred to as organ transplant rejection, and graft-versus-host reaction (GVHR). HVGR is divided into 4 types:hyperacute rejection, accelerated rejection, acute rejection and chronic rejection. At present, problems in clinical transplant rejection therapy remained to be solved. One of them is immunosuppressants such as CsA and FK506 can efficiently control acute rejection, but have poor efficiency for chronic rejection, in view that lack of gnosia on mechanism of chronic rejection devolpment and its pathology. One the other hand, 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. Therefore, it is significant to study the mechanism of immunosuppressive drugs on rejection for controlling of chronic graft rejection and maintaining long-term survival of great.Rejection after organ transplantation is the essence of the immunity system against the donor graft antigens in the immune response. After allogeneic organ graft operation, the passenger leukocytes in allograft are primarily mature DC and macrophages and other antigen presenting cells (donor antigen-prsenting cells, dAPC). These cells may enter to the blood circulation or in part lymphoid tissue when donor blood vessels were connected to blood vessels, and directly submit the surface MHC molecules or antigenic peptide/ MHC molecule complexes (pMHC) to the recipient allogeneic T cells (mainly CD8+ T cells and CD4+ T cells) by direct recognition mechanism; The donor antigen (the same kinds of MHC molecules fragment)/recipient MHC molecule complexes from graft exfoliated cells or MHC antigen after processing by APC were submitted to the recipient CD4+ T cells by indirect recognition mechanism to produce immune responses. Activated T cells differentiate into Thl cells and the newly discovered Th17 cells, through secretion of cytokines, chemokines and further activation of CD8+ CTL cells, chemotaxis of macrophages, neutrophils and other inflammatory cells cause inflammation and mediate on graft rejection. Although the mechanism of the immunosuppressant used in clinical could not be coincidence, the same point is that both of them can inhibit the activation, differentiation and proliferation of the T lymphocyte. In recent years, a large number of studies have found that a group of content little CD4+CD25+Foxp3+T cells (Treg cells), as an important immune regulatory function of T cells to mediate immune response in a proactive manner, played an extremely important role in the regulation of maintenance immune tolerance to exogenous antigens or their own. Therefore, it is very important to study the negative regulation mechanism of Treg cells in control of transplant rejection and the dose of immunosuppressant drugs. In particular, studies of the immunosuppressive drugs widely used in clinical such as CsA, FK506, Rapamycin (Rapa) on Treg cells is for guiding the clinical usage of immunosuppressive agents and more effectively control of rejection significant.Rapa first discovered is only used as a low-toxicity antifungal. After immunosuppressive effect was found in 1977, Rapa began trial in the treatment of organ transplant rejection in 1989. From the effects of the present animal experiments and clinical application, Rapa is a highly efficient and low toxicity of new immunosuppressants. The latest research shows that Rapa can promote the generation of Treg cells, but the molecular mechanisms are not yet clear. To study the molecular mechanisms of Rapa that effects on the differentiation and proliferation of CD4+CD25+ Treg cells will provide the basis of experimental data for guiding clinical treatment and reducing the use of immunosuppressive agents and efficiently controling of rejection to improve the survival of organ graft.Part one: The influence of Rapa on the expression of Foxp3 and TGF in CD4+CD25+ T cells in vitroObjective:To observe the proliferation and expression of Foxp3, TGF-βand explore the relationship between TGF-βand the expression of Foxp3 in CD4+CD25+ T cells when Rapa and CD4+CD25+ T cells co-cultured in vitro. Methods:Take spleen from C57BL/ 6 mice under sterile conditions, isolate mononuclear cells and separate CD4+CD25+ T cells by immunomagnetic beads, divide into control group, stimulated group, Rapa group and CsA group; Cells are stimulated with anti-CD28, anti-CD3 and low-dose IL-2 for 72h under, then detect the proliferation of CD4+CD25+ T cells and the expression of Foxp3 by the flow cytometry, the expression of Foxp3 mRNA by Semi-quantitative RT-PCR and the Foxp3 by and ELISA, Results:The proliferation of CD4+CD25+ T cells and the average fluorescence intensity of Foxp3 in Rapa group have more obviously was significantly higher than the stimulated group by FACS detecting; howere, these indicators in CsA group were lower then the stimulated group. Our results suggest that Rapa can promote the proliferation and the expression of Foxp3 in CD4+CD25+T cell, but CsA inhibits these functiones. To further test the cytokines expression, we found that the CD4+CD25+ T cells in Rapa group express TGF-βmRNA and protein were significantly higher than that stimulated group (P<0.01); To contrast, the CD4+CD25+T cells in CsA group express TGF-βmRNA and protein were were lower than the stimulation (P<0.05). The results show that Rapa can promote the expression and the secretion of TGF-βin CD4+CD25+ T cell, while CsA inhibit the expression and the secretion of TGF-βin CD4+CD25+ T cell. Conclusion:Rapa could induce the CD4+CD25+ T cell proliferation and the Foxp3/TGF-βexpression in vitro; the results documented that the effect that Rapa promote CD4+CD25+ T cells proliferation and the Foxp3 expression may be related to the expression and secretion of TGF-β.Part two:TGF-βblocking reduces Rapa induced Foxp3 expression in CD4+CD25+ T cellsObjective:To observe the expression of Foxp3 in Rapa-treated CD4+CD25+ T cells when blocking TGF-P by TGF-βneutralizing antibody and smad3-deficient mice, study the effect of TGF-βon the expression Foxp3 in Rapa-treated CD4+CD25+ T cells. Methods: Take spleen from C57BL/6 mice under sterile conditions, isolat mononuclear cells, separate CD4+CD25+ T cells by immunomagnetic beads, divide into stimulated group, Rapa group and CsA group, Rapa plus anti-TGF-βgroup; Cells are stimulated with anti-CD28, anti-CD3 and low-dose IL-2 for 72h under, then detect the expression of Foxp3 mRNA by Semi-quantitative RT-PCR and the expression of Foxp3 in CD4+CD25+ T cells by flow cytometry.Take spleenic CD4+CD25+ T cells in smad3-/- mice or WT mice under sterile conditions, stimulate with Rapa, anti-CD28, anti-CD3 and low-dose IL-2 for 72h, then detect expression the Foxp3 by Semi-quantitative RT-PCR and ELISA.Results:The expression of Foxp3 mRNA in Rapa group was significantly higher than that in the stimulated group (P<0.01); but that in Rapa plus anti-TGF-βgroup was no significant difference (P>0.05), indicating that the induction of Rapa on Foxp3 was inhibited by TGF-P neutralizing antibodies; and the fluorescence intensities of Foxp3 in each group are respectively to 87.24% in stimulated group,93.62% in Rapa group, 47.36% in Rapa and anti-TGF-P group, suggesting that blocking TGF-βcan inhibit Rapa to promote the expression of Foxp3.The expression of foxp3 mRNA and protein in the spleenic CD4+CD25+ T cells from smad3-/- mice in Rapa treatment was significantly lower (P<0.01)than those of wild-type control group, suggesting that blocking TGF-βsignaling pathway can inhibit the Rapa on CD4+CD25+ T cells to promoe the expression of Foxp3.Conclusion:Blocking TGF-βcan inhibit Rapa-induced the expression of Foxp3 in CD4+CD25+ T cells in vitro; The effect of Rapa on to promotion of the expression of Foxp3 in CD4+CD25+ T cells may be related to induce the expression and secretion of TGF-p.Part three: Rapa promotes CD4+CD25+Foxp3+ T cells generation in hearttransplantation mouse through TGF-signaling pathwayObjective:To further explore the effect of Rapa on the transplant rejection and the influence of TGF-signaling pathway on the relationship between Rapa and the expression of Foxp3 in CD4+CD25+ T cells after allogeneic heart transplantation. Methods:After performing cervical heterotopic heart transplantation with allograft heart from the male BALB/C mice, C57BL/10 recipients were randomly divided into 4 groups (n=6): Normal group, Control group, Rapa group (1.5mg/kg/d) and CsA group (20mg/kg/d) are observed the graft survival time and the pathological changes. After 14 days, the ratioes of CD4+CD25+ T cells in thymus, spleen and lymph node and Foxp3 expression were detected by flow cytometry. The expression of TGF-βmRNA in spleenic CD4+CD25+ T cell are detected by semi-quantitative RT-PCR and the peripheral blood were collected at different time points to determine TGF-βconcentration by ELISA. Results:After 14 days, the ratioes of CD4+CD25+T cells as well as the Foxp3 expression of Treg cells in thymus, spleen and lymph node of Rapa treatment mice were significantly higher than that in CsA treatment group or control group. The TGF-P mRNA expression in spleenic CD4+CD25+T cells were consistent with the changes of the popularation of CD4+CD25+ T cells as well as the expression of Foxp3. The serum level of TGF-P in Rapa treatment group was not only significantly higher than that of CsA treatment group, but also significantly higher than that of control group. The results show that Rapa can promote the proliferation of CD4+CD25+ T cells, the expression of foxp3 and the secretion of TGF-P in allograft mice. In contrast, CsA not only inhibits the proliferation of CD4+CD25+ T cells and the expression of foxp3, but also inhibits the secretion of TGF-β. The heart allograft survival time of Rapa treatment group were significantly prolonged (20.17±3.31 days) compared with the control group(6.67±0.81 days), and compariable with that of CsA treatment group (21.33±4.17 days). The results suggest that Rapa could inhibit graft inflammation and significantly prolong the allograft survival time. Conclusion:Rapa prolong allograft survival related to promote the generation of CD4+CD25+ T cells and the expression of Foxp3 through TGF-βsignaling pathway. |
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