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目的探讨Micro RNA-126对CD4~+Foxp3~+Tregs的外周诱导调控及功能的影响。方法分选Balb/c小鼠脾脏CD4~+CD25-初始T细胞,在anti-CD3/CD28的激活下,用TGF-β进行诱导培养,在第3、5天FACS检测CD4~+Foxp3~+Tregs的比例,Real-time PCR检测mi R-126的表达;采用mi R-126抑制剂抑制mi R-126后,FACS检测CD4~+Foxp3~+Tregs的比例,Real-time PCR检测mi R-126的表达;进而采用mi R-126 ASO下调CD4~+Foxp3~+Tregs中mi R-126的表达,Real-time PCR检测IL-10和TGF-β的表达,CFSE标记技术分析CD4~+Foxp3~+Tregs免疫抑制功能。结果 mi R-126在活化的Tregs中的表达高于在活化的CD4~+CD25-T细胞中的表达(P<0.05);TGF-β在体外诱导生成Foxp3~+CD4~+T细胞的比例组间差异具有统计学意义(P<0.05),同时在Tregs的诱导过程中,mi R-126的表达上调(P<0.05);CD4~+CD25-T细胞体外瞬时转染mi R-126抑制剂,与对照组比较,Foxp3~+CD4~+T细胞的比例组间差异具有统计学意义(P<0.05),mi R-126抑制剂能有效下调mi R-126的表达(P<0.05);与对照组相比,mi R-126 ASO转染组Tregs中Foxp3、CTLA-4和GITR的表达均降低(P<0.05),且TGF-β和IL-10的m RNA相对表达均降低(P<0.05);CFSE标记细胞增殖实验显示,mi R-126 ASO Tregs组CD4~+CD25-T细胞增殖与Tcon组、Control Tregs组比较,差异具有统计学意义(P<0.05)。结论下调mi R-126的表达能显著削弱CD4~+Foxp3~+Tregs的外周诱导和免疫抑制功能。
Objective To investigate the effect of Micro RNA-126 on the regulation and function of peripheral CD4 ~ + Foxp3 ~ + Tregs. Methods The CD4 ~ + CD25 ~ naive T cells from spleen of Balb / c mice were sorted by TGF-β and activated by anti-CD3 / CD28. CD4 ~ + Foxp3 ~ Tregs, Real-time PCR to detect the expression of mi R-126. The percentage of CD4 ~ + Foxp3 ~ + Tregs was detected by FACS after mi R-126 was inhibited by mi R-126 inhibitor. Real- The expression of IL-10 and TGF-β in CD4 ~ + Foxp3 ~ + Tregs was downregulated by mi R-126 ASO. The expression of IL-10 and TGF-β was detected by Real-time PCR and the expression of CD4 ~ + Foxp3 ~ + Tregs immunosuppressive function. Results The expression of mi R-126 in activated Tregs was higher than that in activated CD4 ~ + CD25-T cells (P <0.05). The percentage of Foxp3 ~ + CD4 ~ + T cells induced by TGF- The difference between the two groups was statistically significant (P <0.05). Meanwhile, the expression of mi R-126 was up-regulated during the induction of Tregs (P <0.05). The transient transfection of miR-126 by CD4 ~ + CD25- (P <0.05). Compared with the control group, mi R-126 inhibitor could effectively down-regulate the expression of mi R-126 (P <0.05), and the ratio of Foxp3 ~ + CD4 ~ + T cells in the control group was statistically significant ; Compared with the control group, the expressions of Foxp3, CTLA-4 and GITR in Tregs of mi R-126 ASO transfection group were decreased (P <0.05), and the relative expression of m RNA of TGF-β and IL- P <0.05). The CFSE-labeled cell proliferation assay showed that the proliferation of CD4 ~ + CD25-T cells in the mi R-126 ASO Tregs group was significantly different from that in the Tcon group and Control Tregs group (P <0.05). Conclusions The down-regulation of mi R-126 expression significantly attenuates peripheral induction and immunosuppression of CD4 ~ + Foxp3 ~ + Tregs.