| PartⅠ The effects and mechanisms of SOCS3gene transfection in chronic rejection of the allograft in mouse aorta transplantation modelOjective:To establish the mouse model of allograft aortic transplantation simulated the pathological processes of CAV. And to investigate the effect and mechanism of adenovirus vector mediated SOCS3gene transfection in chronic rejection of allograft in this model.Methods:The mouse model of allograft aortic transplantation, in which aorta of Balb/c was transplanted into C57b1/6, was developed. The recipients were randomly assigned to3groups:1. the control group,2. Ad-SOCS3group,3. Ad-GFP group.24hours after operation,100μl sterile PBS injection,1×109pfu the recombinant adenovirus vector carrying mouse SOCS3gene (Ad-SOCS3) and1×109pfu vector only carrying GFP reported gene (Ad-GFP) were injected by tail vein, respectively. Three mice of each group were sacrificed in the1st,3rd,5th,7th,14th days after that, and RT-PCR was used to detect the SOCS3mRNA of the allograft, compared with normal aorta of C57bl/6mice. Another three mice were sacrificed to determine the effect of transfection about allograft, liver, spleen and heart, by detecting the fluorescent light with fluorescent microscope, in the3rd day. Nine mice of each group were sacrificed in the8th weeks. The allografts were used for RT-PCR and Western blotting to detect the mRNA and protein expression of SOCS3, STAT3, IFN-y, T-bet, IL-10, GATA-3, iNOS, CXCL-10, Arg-1, Fizzl, VCAM-1, and for Western blotting to detect the protein expression of P-STAT3, and for histological and immunohistochemical analyses. The measurement of stenosis rate of the allograft was performed in hematoxylin-eosin staining section. The macrophages and CD4-positive cells were counted in CD11b and CD4immunohistochemical section. Flow cytometry were used to detecte the rates of CD4+T-bet+/monocyte and CD4+GATA-3+/monocyte in spleen.Rresults:1.Compared with the control group and Ad-GFP group, SOCS3mRNA expression in Ad-SOCS3group can be significantly enhanced in the allograft from the first to the fourteenth day, and were up-regulated from the first day, and peaked on the fifth day, and down-regulated after that.2. The neointimal hyperplasia in the allograft was observed. Compared with the control group and Ad-GFP group, the stenosis rate of the allograft in Ad-SOCS3group can be significantly decreased.3. Inflammatory cell infiltration in the allograft was observed. Compared with the control group and Ad-GFP group, the number of macrophages and CD4-positive cells in the allograft in Ad-SOCS3group can be significantly decreased.4. Compared with the control group and Ad-GFP group, SOCS3, IL-10, GATA-3, Arg-1, Fizzl mRNA and protein expression of the allograft in Ad-SOCS3group were significantly up-regulated, and STAT3, IFN-γ, T-bct, iNOS, CXCL-10, VCAM-1mRNA and protein expression as well as P-STAT3protein expression of the allograft in Ad-SOCS3group were significantly down-regulated.5. Compared with the control group and Ad-GFP group, the rates of the CD4+T-bet+/monocyte in Ad-SOCS3group were significantly down-regulated, and the rates of the CD4+GATA-3+/monocyte in Ad-SOCS3group were significantly up-regulated.Conclusion:These results imply that the rejection of the allograft in the recipients took place when the mouse model of allograft aortic transplantation was established. SOCS3gene transfection by the recombinant adenovirus vector carrying mouse SOCS3gene can be efficient and up-regulate SOCS3mRNA and protein expression in the allograft. Furthermore, over-expression of SOCS3in allograft can inhibit neointimal hyperplasia and down-regulate the stenosis rate, which indicates the inhibition of chronic rejection of allograft in mice aorta transplantation model. This effect could be caused by inhibited activation of JAK/STAT pathway, relieving the systemic and local immune response, and then reducing macrophages and CD4+Th cells infiltration, pro-inflammatory cytokines expression, as well as increasing anti-inflammatory cytokines expression in allograft. The effect and mechanism of SOCS3gene in the immunoreaction of mouse macrophages and Th cells may need a further research. PartⅡ The effects and mechanism of SOCS3gene transfection in the immunoreaction of mouse macrophages and Th cellsObjective:Using adenovirus vector mediated SOCS3gene transfection to intervene LPS-induced macrophages polarization, and PHA-induced Th cells differentiation, as well as the expression of cytokines in vitro. To investigate the effect and mechanism of SOCS3gene in the immunoreaction of mouse macrophages and Th cells, which are the main effector cells in chonic rejection.Methods:Both of the macrophages and CD4+Th cells from C57bl/6mouse were isolated and cultured, and divided randomly into three groups:1. the control group,2. Ad-SOCS3group,3. Ad-GFP group. Sterile PBS, recombinant adenovirus vector carrying mouse SOCS3gene (Ad-SOCS3) and vector only carrying GFP reported gene (Ad-GFP) were transfected into cells, respectively. LPS were used to induce the macrophages polarization for24hours, and PHA were used to induce the Th cells differentiation for48hours. RT-PCR and Western blotting were used to detect the mRNA and protein expression of SOCS3, STAT3, iNOS, TNF-α, IL-6, CXCL-10, Arg-1, Fizzl, CCL-22, TGF-β of macrophages, and SOCS3, T-bet, IL-2, IFN-y, STAT4, IL-12Rβ2, GATA-3, IL-4, IL-6, IL-10, STAT6of Th cells. Western blotting was used to detect the protein expression of P-STAT3of macrophages. IFA were used to detecte the iNOS, Arg-1, IFN-y and IL-10positive cells. Flow cytometry were used to detecte the CD86, CD80, T-bet+and GATA-3+positive cells.Results:1.The appropriate effect can be achieved by using adenovirus vector mediated SOCS3gene transfection with the condition that MOI equal to80, for48hours in macrophages, and MOI equal to160, for72hours in CD4+Th cells.2. Compared with the control group and Ad-GFP group, the expression of mRNA and protein about SOCS3, Arg-1, Fizzl, CCL-22, TGF-β of the macrophages and SOCS3, GATA-3, IL-4, IL-6, IL-10, STAT6of CD4+Th cells in Ad-SOCS3group were significantly up-regulated, and that about STAT3, iNOS, TNF-a, IL-6, CXCL-10of the macrophages and T-bet, IL-2, IFN-γ, STAT4, IL-12Rβ2of CD4+Th cells in Ad-SOCS3group were significantly down-regulated. The expression of protein about P-STAT3of the macrophages Ad-SOCS3group was significantly down-regulated too.3. Compared with the control group and Ad-GFP group, the expression of iNOS, IFN-γ in Ad-SOCS3group were significantly down-regulated in IFA, but the expression of Arg-1, IL-10were significantly down-regulated.4. Compared with the control group and Ad-GFP group, the rate of the CD86, T-bet positive cells in Ad-SOCS3group was significantly down-regulated, and the rate of the CD80, GATA3positive cells in Ad-SOCS3group was significantly up-regulated.Conclusion:The results of our reseach imply that SOCS3gene transfection by the recombinant adenovirus vector carrying mouse SOCS3gene can inhibit the LPS-induced M1macrophages polarization and PHA-induced Thl cells differentiation as well as the ability of pro-inflammatory, in the meantime, can promote M2macrophages polarization and Th2cells differentiation as well as the ability of anti-inflammatory. It was on account of down-regulation of the JAK/STAT pathways by the overexpression of SOCS3that M1macrophages polarization and Thl cells differentiation were inhibited. Furthermore, the inhibition of JAK/STAT pathways and down-regulation of some pro-inflammatory cytokines expression, could alleviate the feedback inhibition on M2macrophages polarization and Th2cells differentiation. These results in vitro confirm that SOCS3gene may play an alleviating role in the development of chronic rejection of allograft in mouse aorta transplantation model, by regulating macrophages polarization, Th cells differentiation, and the expression of cytokines. These data can provide a novel clinical therapeutic strategy with CAV after heart transplantation. |
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