| Part Ⅰ Effect of BMSCs on DCs phenotype and detection of CD45RB~+ DCs immune function in ratsObjective: To study the effect of rat bone marrow mesenchymal stem cells on the phenotype of dendritic cells derived from allogeneic spleen, and the immune status of the affected CD45RB~+ DCs.Methods: SD rat BMSCs and allogeneic dendritic cells were cultured in vitro for 5days in the same cell proportion. According to the effect of BMSCs on dendritic cells mechanism, the mice were divided into control group and experimental group, control group and the experimental groups were set up: group A: DC with LPS co cultured, group B: BMSC and dendritic cells co cultured. The changes of surface molecular phenotype of dendritic cells(ILT4, PDL1, CD1 d, CD45 RA, CD45 RB, CD14, CD80, CD86, MHC-II)before and after co culture were analyzed by flow cytometry, and the morphological changes of DC cells were also analyzed.CD45RB~+DCs induced by BMSCs cells were isolated by flow cytometry. The experimental group were divided into three groups: group A: immature DC group, B group:CD45RB~+DCs group, C group: mature DC group. Were used to detect the cell costimulatory molecules(CD80, CD86 and MHC) expression and qPCR method and each group DC antigen uptake of bead-OVA complexes uptake measured the expression of Foxp3 mRNA in different DC cells and Western bloting to detect Foxp3 protein expression.Results: Compared with the control group, BMSC co cultured group of the dendritic cells ILT4 and CD45 RB expression was significantly increased, and MHC-II and CD86 expression was significantly down regulated. CD45RB~+ DCs surface low expression of costimulatory molecules CD80, CD86 and MHC, imDC feature; CD45RB~+ DCs group andimDC group uptake was significantly lower than that of the MDC group, CD45RB~+ DCs group and group imDC with Foxp3 mRNA and the expression of Foxp3 expression were significantly higher than those of mDC group.Conclusions: BMSC can up regulate the expression of CD45 RB and ILT4 on the surface of DC cells, and down regulate the expression of MHC2 and CD86, especially with the most significant change of CD45 RB. The CD45RB~+ DCs induced by BMSC cell population uptake ability is low, the expression of mRNA FOXP3 and Foxp3 protein was significantly up-regulated, showing the characteristics of the immune behavior of imDC.MSCs may inhibit the maturation of CD45RB~+ DCs through the expression of Foxp3,which has an effect on the immune regulation of CD45RB~+ DCs.Part Ⅱ Study of the effect of CD45RB~+ DCs on the function of T cells in vitroObjective: To detect the effect of different DCs cells on the proliferation of T lymphocytes and their effects on T lymphocyte subsets by co culture of DCs cells and T lymphocytes in vitro.Methods: The CD4+T cells were co cultured with DC and SD rats in Wistar rats,including the following groups. Group A: con group as negative control group, the SD rats of CD4 + T cells, group B: ConA as positive control group, namely separation of SD rats after CD4 + T cells given ConA stimulation(ng / ml), group C: in ConA positive control group based on adding Wistar rat immature DCs cells were co cultured DC:T=1:10, D group: on ConA positive control group based on adding Wistar rats CD45RB~+DCs cells were co cultured DC:T=1:10 E group: in ConA positive control group based, with Wistar rat mature DCs cells were co cultured DC:T=1:10.Co culture after the completion, CD4 + T cells of each function was determined, including CCK-8 assay to detect T cell absorbance value to understand the proliferation of T cells; RNA was extracted with Th0 cells detected by qPCR method to the expression of Th1, Th2, Th17 and Treg differentiation specific transcription factor T-bet, GATA-3, ror gamma t, Foxp3. The number and ratio of CD4+Foxp3+T cells in each group were detected by flow cytometry. The expression of various cytokines in the supernatant of co culture system(IL-2, IL-4, IL-10, IL-17 A, CBA,IFN-, TNF-, IL-6) was detected in the supernatant of co culture system.Results: cells were cultured, the CD45RB~+DCs group T cell proliferation abilitydecreased significantly, group have significant difference; while increasing the number of CD45RB~+DCs of CD4 + Foxp3 + T cells, and control group have significant difference;CD45RB~+DCs group and imDCs specific transcription factor T-bet, Foxp3 upregulation,GATA-3, ror gamma downregulated, IL-2 and IFN- levels decreased gradually, IL-4,IL-10 and TGF- 1 levels gradually increased, and control group have statistical difference.Conclusions: CD45RB~+DCs in vitro can inhibit proliferation of reactive T lymphocytes, and increase of CD4 +Foxp3 + T cell number and ratio, and increased TGF beta 1, IL-4, IL-10 levels and decreased IL-2 and IFN- levels and promote th cell differentiation to Th2 shift and other factors.Part Ⅲ Experimental study on the induction of allograft immune tolerance by intravenous infusion of CD45RB~+DCsObjective: To study the CD45RB~+DCs cell transplantation in rats induced by pretreatment method of cardiac allograft immune tolerance in rats.Methods:Wistar rats to establish SD rat allograft heterotopic cervical heart transplantation model. In the 1 weeks prior to heart transplantation, intravenous injection of SD receptor in rats. Experimental groups: group A(control group): Wistar rats and SD rat abdominal heterotopic heart transplantation; group B(imDCs injection group): receptor(SD rat) via tail vein injection of donor(Wistar rats) mature dendritic cells(DC) 1 x106/0.2ml; group C(CD45RB~+ DC cell injection group): receptor(SD rat) via tail vein injection of donor(Wistar rats) CD45RB~+ dendritic cells(DC) 1 x 106/0.2ml; D group(MDCs injection group): receptor(SD rat) via tail vein injection of donor(Wistar rats)cells of MDCs to 1 x 106/0.2ml. Palpation method to observe the survival time of the cardiac allograft. After transplantation, first, 3, 5 days respectively from cardiac allograft lymphocytes were isolated, RNA extraction and relative quantitative RT-PCR was used to detect IL-2, IL-4, IL-10, miR-7a, miR-155, IFN- gamma, miR-182, miR-183,miR-434-3p. Were collected from peripheral blood by CD4+ Foxp3+T lymphocytes by flow cytometry.Results: after allograft of rat heart. CD45RB~+DCs group and imDCs shift plant survival time than in the control group was significantly prolonged, a statisticallysignificant difference, gradually increase the proportion of CD4 + Foxp3 + T lymphocytes,the 5 day performed significantly, a statistically significant difference. After transplantation, first, 3, 5 days miR-7a, miR-182, miR-183 level increased, miR-434-3p level decreased gradually, each time point had significant difference, but at the same time,some groups have no obvious difference. Third days and fifth days CD45RB~+DCs group and imDCs group miR-155 levels decreased, compared with the control group. On the fifth day after transplantation, IL-2 and IFN- levels were significantly decreased and elevated levels of IL-4, compared with the control group CD45RB~+DCs group and imDCs group with significant difference; elevated CD45RB~+DCs IL-10 levels. There was significant difference.Conclusions: Rat cardiac allograft, miR-155 receptor, miR-182, miR-183 and miR-7expression increased, miR-434-3p expression decreased. Vein were injected with donor CD45RB~+DCs and imDCs cells in can increase by in vivo CD4 + Foxp3 + regulatory T cell number and reduced receptor in rat miR-155 expression level, improve graft cytokine IL-4,IL-10 expression level decreased IL-2, IFN- expression levels prolong the cardiac allograft survival time. |
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