目的 rhG-CSF既是造血干细胞动员剂,也是免疫耐受诱导剂,可减轻移植物抗宿主病(GVHD);干扰素调节因子8(IRF8)拮抗小鼠RORγt的作用,可以抑制小鼠初始T细胞向Th17细胞分化;本文旨在研究rhG-CSF动员对IRF8基因表达及蛋白水平、外周血Th17和Treg细胞的比例和Th17分化的负性调控因子IL-27的调节作用.方法 采集10名造血干细胞移植供者rhG-CSF动员前后(第5天)外周血,通过免疫磁珠法分离CD4+T细胞,qRT-PCR及Western blotting检测CD4+T细胞IRF8基因及蛋白水平,流式细胞术检测Th17(CD3+CD8-IL-17+)和Treg细胞(CD4+CD25+Foxp3+)比例,ELISA法检测血清中IL-17及IL-27的浓度.结果 动员前、后外周血外周血单个核中细胞的比例(％):Th17细胞(2.597±1.993)％比(0.355±0.129)％(P＜0.01)),Treg细胞(0.468±0.522)％比(1.491±1.216)％(P＜0.01);动员前、后IL-17A(pg/ml):(21.75±18.77) pg/ml比(15.53±10.89) pg/ml (P＜0.01);动员前、后IL-27(pg/ml):(5 180.06±1 860.75) pg/ml比(3 806.56±1 658.89) pg/ml(P＜0.01).IRF8基因表达在动员后基因表达相对增加1.69倍,IRF8蛋白在动员后比动员前表达显著增加.结论 rhG-CSF抑制供者外周血Th17细胞、促进Treg细胞群分化,诱导免疫耐受,可能与rhG-CSF促进CD4+T细胞中IRF8、IL-27和IRF8负性调控Th17细胞分化有关.%Objective Recombinant human G-CSF(Rh-GCSF) is both an initiator of hematopoietic stem cells and an inducer of immune tolerance,and can reduce graft versus host disease (GVHD).Interferon regulatory factor 8 (IRF8) antagonizes the effect of ROR γt and inhibites the differentiation of mouse naive T cells into Th17 cells.The aim of this study is to investigate the effects ofrhG-CSF mobilization on the gene expression and protein level of IRF8,the proportions of Th17 and Treg cells in peripheral blood,and the role of negative regulatory factor IL-27 of Th17 differentiation.Methods We collected peripheral blood from 10 hematopoietic stem cell transplantation donors before and after the mobilization of rhG-CSF (fifth day).CD4+ T cells were isolated by immunomagnetic beads.IRF8 mRNA and protein in CD4+ T cells were detected by qRT-PCR and western blotting,respectively.The percentages of Th17 (CD3+ CD8-IL-17) and Treg cell (CD4+ CD25+ Foxp3+) were measured by flow cytometry.The concentrations of IL-17 and IL-27 in serum were detected by ELISA.Results The percentages of mononuclear cells in peripheral blood before and after mobilization were as below:Th17 cells (2.597±1.993)％ vs.(0.355±0.129)％ (P ＜ 0.01);Treg cells (0.468±0.522)％ vs.(1.491±1.216)％ (P ＜ 0.01).In addition,the cytokine concentrations in serum before and after mobilization were as below:IL-17A (21.75±18.77) pg/ml vs.(15.53±10.89) pg/ml (P ＜ 0.01);IL-27 (5 180.06±1 860.75) pg/ml vs.(3 806.56±1 658.89) pg/ml (P ＜ 0.01).The expression of IRF8 mRNA increased by 1.69 folds,and the expression of IRF8 protein was significantly higher after than before the mobilization.Conclusions RhG-CSF inhibits Th17 cells,and promotes the differentiation of Treg cells in donor peripheral blood,and induces immune tolerance.It may be related to RhG-CSF promoting IRF8 expression in CD4+ T cells.IL-27 and IRF8 negatively regulate Th17 cell differentiation.
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