Application Of CD4~+CD25~+T Regulatory Cells In Trachea Allogratf Transplant Model Of Mice To Reject Anti-gratf Response

Objective：Establish a method of cultivation and purification of dendritic cells (DCs)from mouse bone marrow in vitro to provide experimental material andestablish basis for the study of immunological tolerance. Immature dendriticcells (iDCs) can induce autoimmune tolerance by activation of antigen-specificCD4~+CD25~+Treg cells. To study the role of CD4~+CD25~+Treg cells in anti-graft rejection response and potential value in application of lung tran-splantation.Methods:Extracted the BALB/c mice spleen lymphocytes and bone marrow cellsunder sterile conditions, and cultured DCs in co-induced of recombinantgranulocyte-macrophage colony-stimulating factor (GM-CSF) andinterleukin (IL-4). Then, the changes of DC morphology were observed underlight microscopy and CD80, CD86expression levels were detected with flowcytometry. BALB/c mice were trachea allograft recipients and C57BL/6J micewere used as donors. To study the role of CD4~+CD25~+Treg cells in anti-graftrejection response and potential value in application of lung transplantation, weestablished mice trachea allograft transplant model and sort CD4~+CD25~+Tregcells from mice spleen lymphocytes by MACS. FCM assays were done toanalyze the ratio of marker protein, Foxp~3, expression in CD4~+CD25~+Tregcells. Alveolar cells were used as a non-specific antigen to activate iDCs intomDCs, and co-cultured with CD4~+CD25~+Treg cells to activate Treg cells. Theactivated Treg cells were inject into mice with trachea allograft transplantation to reduce rejection response. The CD4~+CD25~+Treg cells (2×10~5cells) ofBALB/c mice, alveolar cells (1×10~5cells) of C57BL/6J and DC cells (1×10~5cells) of BALB/c mice were mixed and cultured at37℃in5%CO2. After3days, CD4~+CD25~+Treg cells can full activation in vitro, injected into the micevena caudalis. The allografts were extracted and done pathological examinationwith H&E staining.Results:Bone marrow cells appeared irregular and formed dendritic processes after3days cultured with stimulating factor in vitro which showed the typicalmorphology of dendritic cells, and co-stimulatory molecules (CD80, CD86)were low expression on the cellular surface. The amount of DC from mice bonemarrow was rare. Usually it should be induced by GM-CSF and IL-4, and thecultured DCs were gradually proliferative after3days. Every2mice bonemarrow cells can obtain2.0~5.0×107DC, which can meet the need of theexperiment. CD80, CD86can be detected in BM cells which were induced byGM-CSF and IL-4in the third day, while, they were in low expression. In thefifth day, the percentage of CD80expression was12times more than it in thefirst3days. While, there was no significant change of the percentage of CD86expression compared with the3rd day. In the7th day, the percentage of CD80expression was3times more than it in the5th days, while, CD86was also inlow expression. The results show that with the prolongation of cultural time,the percentage of CD80expression was increased. The purity of CD4~+CD25~+Treg cells were over80%and specifically express Foxp3. when added thecytokines IL-10into the culture,the proliferation of Treg was accelerated. eachcase model of the experiment costs average elapsed time (10±5) minutes tocollect from the donor and transplant. Trachea tissue sections were stained withH&E for pathological examination. Staining results for Group1, no tracheaallograft transplantation, showed that3layers of trachea wall can be seen clearly: mucosa, submucosa and adventitia; Staining results for Group2, CD4~+CD25~+Treg cells administration for7days, showed that3layers structure ofthe trachea still exist, and small amount of inflammatory cells can be seen ontracheal center; Staining results for Group3, CD4~+CD25~+Treg cellsadministration for9days, showed that most mucosa was absent, submucosaand adventitia of trachea structures still exist, and tracheal center also containedsmall amount of inflammatory cells; Staining results for Group4, salineadministration for7days, showed that mucosa and submucosa of trachea wereabsent, and only adventitia of trachea exist; Staining results for Group5, salineadministration for9days, showed that mucosa, submucosa and adventitia oftrachea were almost dissolved, and tracheal center was eroded by inflammatorycells.Conclusion:1. Compared with the spleen cells, bone marrow cells are not only rich inDC precursor cells but also induced into DCs with a short time.2. Tracheaallograft transplant in mouse as a model of allograft rejection after lungtransplantation displays many advantages, with small operative wound, strongstability, and well repeatability.3. cytokines IL-10is capable of promoting Tregcells proliferation.4. Our research imply that CD4~+CD25~+Treg cells play anessential role in reducing rejection, and lay the foundation for reject anti-graftresponse in clinical application.