目的:探讨马尔尼菲青霉菌(TM)对树突状细胞(DCs)分化成熟及对DCs诱导CD4+T淋巴细胞向Th17/Treg分化平衡的影响.方法:诱导小鼠骨髓来源的单个核细胞增殖分化为DCs,观察TM刺激后的DCs的形态,流式细胞仪检测DCs表面共刺激分子,酶联免疫吸附试验(ELISA)法检测培养上清液中的白介素(IL)-6及转化生长因子(TGF)-β浓度.从小鼠脾脏分离出CD4+T细胞,将TM与DCs及CD4+T细胞共培养24 h后,流式细胞术观察各组细胞Th17及Treg的阳性表达率,实时荧光定量PCR(qPCR)检测各组RORγt mRNA和Foxp3 mRNA表达,ELISA法检测各组培养细胞上清液IL-17A、IL-10及TGF-β的水平变化.结果:DCs在与TM共培养24 h后可见大量DCs中含TM.TM可上调共刺激分子CD40、CD80、CD86和细胞因子IL-6、TGF-β表达(P＜0.05).CD4+T细胞联合经TM刺激成熟后的DCs时,可使Th17阳性表达率降低而Treg阳性表达率升高(均P＜0.05),Th17/Treg平衡倾向于Treg,且Treg可分泌大量的IL-10及TGF-β发挥免疫抑制作用.结论:TM可诱导DCs的成熟,经TM刺激成熟的DCs可诱导CD4+T淋巴细胞向Th17/Treg分化平衡,从而发挥免疫抑制效应,这可能是TM逃避宿主免疫,在体内存活致病及复发的机制.%Objective:To investigate the effects of P.marneffei on dendritic cells (DCs) maturation and modulation of Th17/Treg cell balance.Methods:Mononuclear cells derived from mice bone marrow were differentiated into DCs.The morphology of DCs after P.marneffei stimulation was observed.The expressions of costimulatory molecules on the surface of DCs were detected by flow cytometry.The concentrations of interleukin (IL)-16 and transforming growth factor (TGF)-β in supernatants were measured by enzyme-linked immuno sorbent assay (ELISA).The CD4+ T cells were isolated from mice spleen with magnetic activated cell sorting technique.P.marneffei was co-cultured with DCs and CD4+T cells for 24 hours,and the percentages of Th17 and Treg were analyzed by flow cytometry.The mRNA expression of ROR γt and Foxp3 was determined by qPCR.The levels of IL-17A,IL-22 and TGF-β in supernatants were also detected by ELISA.Results:Numerous P.marneffei yeast cells were observed in co-cultures of DCs and P.marneffei.The positive overexpression rate of CD40,CD80 and CD86 on the surface of DCs as well as IL-6 and TGF-β levels were obviously increased by P.marneffei.The Th17 percentage was decreased,while Treg was increased in matured DCs stimulated by P.marneffei.Moreover,Treg cells could secrete large amounts of IL-10 and TGF-β to exert an immunosuppressive effect.Conclusion:P.marneffei exerts an immunosuppressive effect though inducing DCs maturation and regulating the balance of Th17/Treg on CD4+ T cells.This may be the mechanism by which P.marneffei evades host immunity,surviving and relapse in vivo.
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