【摘要】：目的探討微小核糖核酸(miRNA)-155對(duì)調(diào)節(jié)性T細(xì)胞(Treg)的兩種亞型誘導(dǎo)性Treg(iTreg)和天然性Treg(nTreg)的影響。方法采用健康成人外周血分離獲取的外周血單個(gè)核細(xì)胞(PBMC),利用磁性細(xì)胞分選法分別獲取幼稚T細(xì)胞和nTreg。培養(yǎng)階段將細(xì)胞分為3組:對(duì)照組(幼稚T細(xì)胞加入白細(xì)胞介素-2培養(yǎng))、iTreg組(幼稚T細(xì)胞加入白細(xì)胞介素-2和轉(zhuǎn)化生長(zhǎng)因子-β培養(yǎng))和nTreg組(nTreg加入白細(xì)胞介素-2培養(yǎng))。每組再分為3個(gè)亞組:未處理亞組、scramble亞組和miRNA-155拮抗劑亞組(每亞組3個(gè)孔)。采用低密度芯片分析方法檢測(cè)3組中的未處理亞組細(xì)胞中miRNA-155的基因表達(dá)水平。采用流式細(xì)胞術(shù)檢測(cè)3組中各亞組細(xì)胞的表面標(biāo)志物CD25、Foxp3、CD127水平。采用流式細(xì)胞術(shù)檢測(cè)3組中各亞組細(xì)胞的CD4+CD25+Foxp3+SOCS1+Treg比例;采用流式細(xì)胞術(shù)檢測(cè)3組中各亞組細(xì)胞的Treg抑制功能。結(jié)果與對(duì)照組和iTreg組比較,nTreg組細(xì)胞的miRNA-155表達(dá)水平明顯降低,差異有統(tǒng)計(jì)學(xué)意義(均為P0.05)。與對(duì)照組和iTreg組比較,nTreg組的SOCS1表達(dá)水平明顯升高,差異均有統(tǒng)計(jì)學(xué)意義(均為P0.05)。加入miRNA-155拮抗劑后并未導(dǎo)致Foxp3、CD127和CD25等Treg重要表面標(biāo)志物發(fā)生明顯的變化。與對(duì)照組和iTreg組比較,nTreg組的SOCS1表達(dá)水平明顯升高,差異均有統(tǒng)計(jì)學(xué)意義(均為P0.05)。iTreg組中的未處理亞組細(xì)胞miRNA-155表達(dá)水平較低,而拮抗劑抑制其表達(dá)之后(miRNA-155拮抗劑亞組),能夠使其SOCS1表達(dá)升高。iTreg組中,與未處理亞組比較,miRNA-155拮抗劑亞組的Treg抑制功能在1∶8、1∶16、1∶32的比例時(shí),顯示出了更強(qiáng)的抑制功能(均為P0.05)。結(jié)論體外拮抗miRNA-155對(duì)nTreg的抑制功能無明顯影響,但是能夠增加iTreg的SOCS1表達(dá)水平和體外抑制功能。
[Abstract]:Objective to investigate the effects of (miRNA) 155 on two subtypes of regulatory T cell (Treg), inducible Treg (iTreg) and natural Treg (nTreg). Methods (PBMC), of peripheral blood mononuclear cells isolated from healthy adults was used to obtain immature T cells and nTreg. by magnetic cell sorting method. At the culture stage, the cells were divided into three groups: control group (immature T cells cultured with interleukin-2 (), iTreg) group (immature T cells added interleukin-2 and transforming growth factor- 尾 culture) and nTreg group (nTreg supplemented with IL-2 culture). Each group was subdivided into 3 subgroups: untreated subgroup, scramble subgroup and miRNA-155 antagonist subgroup (3 holes per subgroup). Low density microarray analysis was used to detect the expression of miRNA-155 gene in untreated subgroups of three groups. Flow cytometry was used to detect the level of surface marker CD25,Foxp3,CD127 in each subgroup of the three groups. Flow cytometry was used to detect the proportion of CD4 CD25 Foxp3 SOCS1 Treg in each subgroup of the three groups, and flow cytometry was used to detect the inhibitory function of Treg in each subgroup of the three groups. Results compared with control group and iTreg group, the expression of miRNA-155 in nTreg group was significantly lower than that in control group and iTreg group (P0.05). Compared with control group and iTreg group, the expression of SOCS1 in nTreg group was significantly higher than that in control group and iTreg group (P0.05). The addition of miRNA-155 antagonist did not cause significant changes in Treg surface markers such as Foxp3,CD127 and CD25. Compared with the control group and the iTreg group, the expression of SOCS1 in the nTreg group was significantly higher than that in the control group and iTreg group, and the difference was statistically significant (P0.05) the expression of miRNA-155 in the untreated subgroup was lower than that in the). ITreg group. After the antagonist inhibited its expression (miRNA-155 antagonist subgroup), the SOCS1 expression was increased. In iTreg group, compared with untreated subgroup, the Treg inhibitory function of miRNA-155 antagonist subgroup was 1: 8: 1: 161: 32. It showed stronger inhibitory function (P0.05). Conclusion antagonizing miRNA-155 in vitro has no obvious effect on the inhibitory function of nTreg, but it can increase the SOCS1 expression level of iTreg and its inhibitory function in vitro.
【作者單位】： 南京醫(yī)科大學(xué)第一附屬醫(yī)院肝臟外科;
【基金】：國(guó)家自然科學(xué)基金(81273262、81210108017、81100270、81070380)
【分類號(hào)】：R392

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