The Effect Of Treg Infusion Under Different MiR-146a Expression On The Immune Rejection Of Cardiac Allograft In Mice

ObjectiveTo investigate the effect of miR-146a regulated Tregs-tranfusion on cardiac allograft rejection in mice and to explore the effect of regulating miR-146a expression on Treg cell suppression function.MethodsSeparated the regulatory T cells in the spleen of mice by flow cytometry and proliferated the regulatory T cells by Miltenyi Biotec Reagent in vitro.Transfected the Tregs by miR-146a agomir and miR-146a antagomir to up-regulate or down-regulate the expression level of miR-146a in Tregs.Established four groups of the cardiac allograft mice models,NS control group,normol Tregs group,miR-146a-up-regulated group and miR-146a-down-regulated group.Transfused the corresponding Tregs through the dorsal vein of penis of the mice(the number of cells-infusion is 1×10~6).Observed the of donor heart and the donor heart was sacrificed for pathological analysis on the seventh day after the operation.T cell subsets groups in the recipient spleen in different groups were detected by flow cytometry and the expression level of IFN-gamma,IL-4 and IL-17 was detected by rt-PCR.ResultsThe number of Tregswastentimes multiplied after separation and proliferation and the purity was 92.3%.There was no significant difference in the expression level of miR-146a.Compared with the NS control group,survival time in empty group and miR-146a-up-regulated group was longer(P<0.05),the acute rejection is more mild(P<0.05),the number of CD4~+T cells was significantly decreased(P<0.05),there was no significant difference between the number of CD8~+T cells(P>0.05),the number of Th1,Th2 and Th17 cells was not significantly different(P>0.05),and there was no significant difference between the expression level of IFN-γ,IL-4 and IL-17 in donor heart(P>0.05);while survival time in miR-146a-down-regulated group and the acute rejection grading were not significantly different(P>0.05),the number of CD4~+T cells was significantly decreased(P<0.05),there was no significant difference between the number of CD8~+T cells(P>0.05),the number of Th1 was significantly increased(P<0.05),the number of Th2 and Th17 cells were not significantly different(P>0.05),the expression level of IFN-γwas significantly increased,and there was no significant difference between the expression level of IL-4 and IL-17 in donor heart(P>0.05).Compared with the empty group,survival time in miR-146a-up-regulated group was longer(P<0.05),the acute rejection grading was more mild(P<0.05),the number of CD4~+T cells was significantly increased(P<0.05),there was no significant difference between the number of CD8~+T cells(P>0.05),the number of Th1,Th2 and Th17 cells and the expression level of IFN-γ,IL-4 and IL-17 in donor heart were not significantly different(P>0.05);while survival time in miR-146a-down-regulated group was significantly shorter(P<0.05),the acute rejection grading was significantly higher(P<0.05),the number of CD4~+T cells and CD8~+T cells were no significantly different(P>0.05),the number of Th1 and the IFN-γexpression level were significantly increased(P<0.05),the number of Th2 and Th17 cells and the expression level of IL-4 and IL-17 in donor heart were not significantly different(P>0.05).ConclusionsThe proliferation reagent effectively proliferated the number of Tregs without affecting the expression level of the miR-146a in Tregs in vitro.Tranfusion of Tregs can significantly suppress the acute rejection of cardiac allograft in mice,further,up-regulate the expression level of miR-146a in Tregs can strengthen the suppression function while down-regulate the expression level of miR-146a in Tregs can attenuate the suppression function.