TGF-β1和IL-10对间充质干细胞来源细胞外囊泡免疫调节功能的影响  

Effect of TGF-β1 and IL-10 on the Immunoregulatory Function of Extracellular Vesicles Derived from Mesenchymal Stem Cells

作　　者：马聪 张青宜[1] 郭子宽[3] 王恒湘[1] MA Cong;ZANG Qing-Yi;GUO Zi-Kuan;WANG Heng-Xiang(Department of Hematology,Air Force General Hospital,Beijing 100142,China;Dalian Medical University,Dalian 116044,Liaoning Province,China;Department of Experiment Hematology,Institue of Radiation Medicine,Academy of Military Medicine Sciences,Beijing 100850,China)

机构地区：[1]空军总医院血液科,北京100142 [2]大连医科大学,辽宁大连116044 [3]军事医学科学院放射与辐射医学研究所实验血液学研究室,北京100850

出　　处：《中国实验血液学杂志》2018年第6期1785-1792,共8页Journal of Experimental Hematology

基　　金：首都卫生发展科研专项资助项目(2014-2-5122);首都临床特色应用研究与成果推广项目(Z15110000415016)

摘　　要：目的:探讨TGF-β1和IL-10对间充质干细胞(mesenchymal stem cells,MSC)分泌的细胞外囊泡(extracellular vesicle, EV)免疫调控功能的影响。方法:分离扩增人脐带来源的MSC,分别用TGF-β1和IL-10处理72 h,提取上清中MSC-EV。应用BCA法测定提取EV的总蛋白含量,在透射电子显微镜下观察EV的形态结构,流式细胞术检测EV表面标志物以鉴定MSC-EV。在ConA刺激后的人外周血单个核细胞(PBMNC)培养体系中,加入不同处理方法获得的EV,培养72 h后应用流式细胞仪检测PBMNC的凋亡,以及CD4+CD25+/CD127-调节T(Treg)细胞比例。应用CBA试剂盒和ELISA试剂盒检测培养PBMNC上清以及MSC-EV中IL2,IL4,IL6,IL10,IFN-γ,TNF-α,Th17A和TGF-β1的含量。结果:不同处理后的EV在透射电子显微镜下均呈现特征性杯口状膜样结构,均表达CD9,CD44,CD63及CD81。TGF-β1处理后MSC-EV促PBMNC凋亡的能力明显增强(P <0.01),不同处理后EV均可以提高Treg细胞比例。MSC-EV可以提高PBMNC培养上清中IL-10的含量,TGF-β1处理MSC-EV共培养后的PBMNC上清TGF-β1含量较未处理组明显降低(P <0.05)。TGF-β1处理后的MSC-EV样本IL-6含量显著升高,TGF-β1含量降低。结论:TGF-β1处理后MSC-EV的免疫调控功能发生改变,其意义值得进一步研究阐明。Objective: To investigate the effect of immune regulatory molecules TGF-β1 and IL-10 on the immunoregulatory activities of extracellular vesicles(EV) secreted from mesenchymal stem cells (MSCs).Methods:MSC were isolated from human umbilical cord and expanded, then were treated with TGF-β1 and IL-10 for 72h, and MSC-EVs in supernatants were isolated.The total protein content of all samples was determined by BCA methed.The morphological structure was observed by transmission electron microscopy.The surface markers of MSC-EV were analyzed by flow cytometry.The apoptosis of peripheral blood mononuclear cells(PBMNC) stimulated by ConA and the proportion of CD4+CD25+/CD127- (Treg) cells were detected by flow cytometry after incubation with MSC-EV for 72 h.The CBA and ELISA kit were used to detect the contents of IL2, IL4, IL6, IL10, IFN-γ, TNF-α, Th17A and TGF-β1 in PBMC supernatants and MSC-EV.Results: All the samples showed that the typical cup-shaped membranelike structure was observed by transmission electron microscopy, and CD9, CD44, CD63 and CD81 expressed.After TGF-β1 treatment, the MSC-EV displayed the strongest ability to promote PBMNC apoptosis(P<0.01), and in all the samples the proportion of Treg cells increased.MSC-EV could increase the content of IL-10 in the supernatants of PBMNC culture, the content of TGF-β1 in PBMNC supernatants after MSC treatment with TGF-β1 was lower than that in untreated group(P<0.05).The content of IL-6 in MSC-EV increased significantly after treatment with TGF-β1,and the content of TGF-β1 decreased.Conclusion: TGF-β1 alters the immnomodulatory function of MSC-EV and its underlying mechanisms need to be clarified in further investigations.

关 键 词：间充质干细胞 细胞外囊泡 TGF—β 1 IL-10 免疫调控 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]