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

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。

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

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.