miRNA-155对调节性T细胞表型和功能的影响

Effect of miRNA-155 on phenotype and function of regulatory T cell

目的探讨微小核糖核酸(miRNA)-155对调节性T细胞(Treg)的两种亚型诱导性Treg(iTreg)和天然性Treg(nTreg)的影响。方法采用健康成人外周血分离获取的外周血单个核细胞(PBMC),利用磁性细胞分选法分别获取幼稚T细胞和nTreg。培养阶段将细胞分为3组:对照组(幼稚T细胞加入白细胞介素-2培养)、iTreg组(幼稚T细胞加入白细胞介素-2和转化生长因子-β培养)和nTreg组(nTreg加入白细胞介素-2培养)。每组再分为3个亚组:未处理亚组、scramble亚组和miRNA-155拮抗剂亚组(每亚组3个孔)。采用低密度芯片分析方法检测3组中的未处理亚组细胞中miRNA-155的基因表达水平。采用流式细胞术检测3组中各亚组细胞的表面标志物CD25、Foxp3、CD127水平。采用流式细胞术检测3组中各亚组细胞的CD4+CD25+Foxp3+SOCS1+Treg比例;采用流式细胞术检测3组中各亚组细胞的Treg抑制功能。结果与对照组和iTreg组比较,nTreg组细胞的miRNA-155表达水平明显降低,差异有统计学意义(均为P<0.05)。与对照组和iTreg组比较,nTreg组的SOCS1表达水平明显升高,差异均有统计学意义(均为P<0.05)。加入miRNA-155拮抗剂后并未导致Foxp3、CD127和CD25等Treg重要表面标志物发生明显的变化。与对照组和iTreg组比较,nTreg组的SOCS1表达水平明显升高,差异均有统计学意义(均为P<0.05)。iTreg组中的未处理亚组细胞miRNA-155表达水平较低,而拮抗剂抑制其表达之后(miRNA-155拮抗剂亚组),能够使其SOCS1表达升高。iTreg组中,与未处理亚组比较,miRNA-155拮抗剂亚组的Treg抑制功能在1∶8、1∶16、1∶32的比例时,显示出了更强的抑制功能(均为P<0.05)。结论体外拮抗miRNA-155对nTreg的抑制功能无明显影响,但是能够增加iTreg的SOCS1表达水平和体外抑制功能。

Objective To investigate the effect of micro ribonucleic acid（ miRNA）-155 on two subtypes of regulatory T cell（ Treg） ： induced Treg（ iTreg） and natural Treg（ nTreg）. Methods Nave T cells and nTreg were isolated from peripheral blood mononuclear cell（ PBMC） of healthy donors by magnetic cell sorting. Cells were divided into 3 groups during culture,including control group（ nave T cells were cultured with the presence of interleukin-2）,iTreg group（ nave T cells were cultured with the presence of interleukin-2 and transforming growth factor-β） and nTreg group（ nTreg cells was cultured with interleukin-2）.Each group was divided into 3 subgroups（ none,scramble or miRNA-155 antagomir subgroup,3 wells in each subgroup）. Expression level of miRNA-155 gene of none subgroup in 3 groups was detected by low density chip analysis method. The levels of surface marker CD25,Foxp3,CD127 of each subgroup in 3 groups were detected by flow cytometry. The percentage of CD4^＋CD25^＋Foxp3^＋SOCS1^＋Treg and suppressive function ofTreg of each subgroup in 3 groups were also detected by flow cytometry. Results Compared with control group and iTreg group,the expression level of miRNA-155 was significantly lower and SOCS1 was significantly higher in nTreg group（ all in P 0. 05）. After the addition of miRNA-155 antagomir,no significant change was observed in the important surface markers of Treg like Foxp3,CD25,CD127. Compared with control group and iTreg group,the expression of SOCS1 in nTreg group increased significantly（ both in P 〈0. 05）. The expression level of miRNA-155 of none subgroup in iTreg group was lower. The expression of SOCS1 increased after the miRNA-155 was inhibited by antagomir（ miRNA-155 antagomir subgroup）. In iTreg group,the suppressive function of Treg in miRNA-155 antagomir subgroup was higher than that in none subgroup at the ratio of 1∶ 8,1∶ 16 and 1∶ 32（ all in P 〈0. 05）. Conclusions Antagonism of miRNA-155 in vitro has no significant effect on the suppression function of nTreg,but can increase the SOCS1 expression level and suppression in vitro of iTreg.