摘要
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。
To explore the role of mi R-126 in the induction and differentiation of Foxp3~＋regulatory T cells（Tregs）, CD4~＋CD25-na？ve T cells（Tconv） were negatively isolated from splenocytes of Balb/c mice and stimulatedwith anti-CD3/CD28 antibody in the presence of TGF-β and IL-2 for 3 days, 5 days. Then the proportion of Foxp3~＋cells in CD4~＋T cells was analyzed by FACS and the expression of mi R-126 was detected by Real-time PCR. Tregspurified from splenocytes of Balb/c mice by MACS were transiently transfected with mi R-126 ASO or Scramblecontrol, and then cultured or co-cultured with CD4~＋CD25-T in the presence of anti-CD3/CD28 plus IL-2, then theexpression levels of IL-10 and TGF-β were determined by Real-time PCR assay. The proliferation of CD4~＋CD25-Tcells was analyzed by CFSE. We found that the expression level of mi R-126 in activated Tregs was significantlyhigher than that in activated Tcon（P〈0.05）. The proportion of TGF-β-induced Foxp3~＋CD4~＋T cells wassignificantly different in different groups（P〈0.05）, meanwhile, the expression of mi R-126 was upregulated duringthe process of the induction of Tregs（P〈0.05）, and mi R-126 inhibitor could effectively inhibit mi R-126expression（P〈0.05）. Moreover, the proportion of induced Foxp3~＋CD4~＋T cells in mi R-126 inhibitor transfectedgroup was lower than that in control group（P〈0.05）. Compared with control group, the expression levels of Foxp3,GITR and CTLA-4 and the m RNA of IL-10 and TGF-β were significantly decreased in mi R-126 ASO transfectedgroup Tregs（P〈0.05）. Furthermore, the results of CFSE labeling assay showed that the CFSE＋cells in mi R-126 ASO transfected Tregs group was higher than that in control Tregs group（P〈0.05）. These findings suggested thatdownregulation of mi R-126 could significantly impairethe induction and suppressive activity of periphealCD4~＋Foxp3~＋Tregs.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2016年第5期382-387,共6页
Immunological Journal
基金
国家自然科学基金(30901318)
