不同miR-146a表达条件下Treg细胞体内回输对小鼠心脏移植免疫排斥反应的影响

The effect of Tregs-infusion under different miR-146a expression on the immune rejection of cardiac allograft in mice

目的:本研究探讨调控miR-146a Treg细胞体内回输对小鼠心脏移植免疫排斥反应的影响。方法:流式分选Treg细胞并进行体外扩增。转染试剂分别上调和下调Treg miR-146a表达。建立小鼠心脏移植模型,设立对照组,空白Treg组,miR-146a上调组,miR-146a下调组。移植后回输Treg,观察生存曲线,病理分级,测定受体脾T细胞亚群,RT-PCR检测供心IFN-γ、IL-4、IL-17表达。结果:细胞扩增10倍后miR-146a表达无明显变化,并能有效调控miR-146a表达。回输后空白Treg组(中位生存期11 d)及上调组(中位生存期13 d)生存时间延长,病理分级降低(P<0.05);上调组更为明显(P<0.05);下调组(中位生存期7 d)生存时间缩短,病理分级升高(P<0.05),Th1细胞数量(28.6%±2.7%)及供心IFN-γ表达(1.12±0.11)升高(P<0.05)。结论:体外能有效地分选和扩增Treg细胞,并保证miR-146a的表达。回输Treg明显抑制小鼠心脏移植急性排斥反应,上调组抑制功能增强,下调组抑制功能减弱。

Objective： To investigate the effect of regulated Tregs-infusion on cardiac allograft rejection in mice. Methods： Tregs in the spleen of mice in vitro by flow cytometry were separated and proliferated. The cardiac allograft mice model were built and four groups were established： NS control group, empty group, miR-146a-up-regulated group and miR-146a-down-regulated group. We regulated the expression of miR-146a in Tregs, and transfused the Tregs to the cardiac allograft mice model. The survival curve, pathological grade and the T cell subsets in the recipient spleens in different groups were compared. We also detected the expression of IFN-γ, IL-4, IL-17 in donor heart. Results： The proliferation reagent effectively proliferated 10 times the number of Tregs, and there was no significant difference between the expressions of miR-146a before and after the proliferation （P〉0.05）. Survival time of mice in control group （median survival time： 11 days） and miR-146a-up-regulated group（median survival time： 13 days） was longer than that of miR-146a-down-regulated group（median survival time： 7 days）, and the pathological grade was lower （P〈0.05）; survival time of mice in miR-146a-down-regulated group was shorter than control group and miR-146a-up-regulated group, and the pathological grade was higher （P〈0.05）. The number of Th1 （28.6%±2.7%） and the expression of IFN-γ（1.12±0.11） in donor heart in miR-146a-down-regulated group were higher （P〈0.05）. Conclusion： Tregs can be successfully separated and proliferated without affecting the expression level of miR-146a in vitro. Infusion of Tregs can significantly suppress the acute rejection of cardiac allograft in mice, while the suppression function in up-regulated group is stronger than that of down-regulation group.