脂多糖刺激下骨髓间充质干细胞分泌的外泌体对Ly6C单核巨噬细胞亚群的影响  

Exosomes from LPS treated mesenchymal stem cells regulate proportion of Ly6C monocyte population

作　　者：霍然[1,2,3] 傅小媚 邓赛 林潮金 王萍 秦爱萍 杨翔宇 李晓红[2] 余细勇[1,2,3] HUO Ran;FU Xiao-mei;DENG Sai;LIN Chao-jin;WANG Ping;QIN Ai-ping;YANG Xiang-yu;LI Xiao-hong;YU Xi-yong(School of Pharmaceutical Scienees,Southern Medical University,Guangzhou 510515,China;Guangdong General Hospital,Guangdong Academy of Medical Scienees,Guangzhou 510080,China;Key Lab of Molecular Clinical Pharmacology,School of Pharmaceutic Sciences,Guangzhou Medical University,Guangzhou 511436,China)

机构地区：[1]南方医科大学药学院,广东广州510515 [2]广东省人民医院医学研究中心,广东省医学科学院,广东广州510080 [3]广州医科大学药学院分子临床药理学重点实验室,广东广州511436

出　　处：《中国药理学通报》2018年第7期910-917,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金重点项目(No 81330007,U1601227); 广东省重大科技计划资助项目(No 2014A050503047,2015B020225006)

摘　　要：目的探索炎症刺激下,骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)产生的外泌体对小鼠淋巴抗原6C高表达(Ly6C^(high))单核巨噬细胞和Ly6C^(low)两个亚群比例的影响。方法梯度离心法分离BMSCs,鉴定其形态、表面标记及成脂诱导分化能力。脂多糖处理BMSCs 48h,收集上清并提取外泌体,采用Western blot法、透射电镜以及粒径分析鉴定外泌体。磁珠分选小鼠腿骨中的单核巨噬细胞,与外泌体共培养24 h后,LPS刺激48 h,流式细胞术检测Ly6C亚群变化。ELISA检测细胞上清炎症因子的变化。结果 BMSCs表达CD44、CD90等,不表达CD34、CD45,并有成脂分化能力。外泌体表达CD63,粒径大小为(82.4±3.70)nm,LPS刺激下BMSCs产生较多的外泌体(P<0.01)。与LPS组相比,外泌体可以使Ly6C^(high)单核细胞比例上升(P<0.01),Ly6C^(high)巨噬细胞比例下降(P<0.05),且LPS刺激BMSCs产生的外泌体比正常BMSCs产生的外泌体的作用更加明显(P<0.05)。促炎因子IL-6水平也明显上调(P<0.05)。结论 BMSCs产生的外泌体可以改变Ly6C^(high)单核巨噬细胞亚群比例,为BMSCs的临床治疗提供了新的靶点。Aim To determine the effect of exosomes from lipopolysaccharide-treated human bone marrow mesenchymal stem cells on proportion of Ly6C^（high） and Ly6C^（low） monocytes/macrophages in inflammatory microenvironment.Methods BMSCs were obtained by gradient centrifugation,identified and then treated with lipopolysaccharide for 48 h.The exosomes were purified from conditional medium with or without LPS treatment and identified by CD63 protein using Western blot and transmission electron microscope.The diameters and concentration were detected by Nanoparticle Trafficking Analysis（ NTA）.The monocytes/macrophages were sorted from bone marrow of the mice by magnetic beads.Cells were co-cultured with exosomes for 24 hours,and then treated with LPS for 48 hours.The proportion of Ly6C monocytes/macrophages was detected by flow cytometry.Inflammatory cytokines in cell supernatant were investigated using ELISA.Results BMSCs surface markers CD44,CD90 were positively detected,but CD34,CD45 were not expressed.BMSCs presented adipogenic differentiation ability.Exosomes were positively expressing CD63 protein,and NTA showed that the diameters of exosomes were up to（ 82.4 ± 3.7） nm.BMSCs stimulated by LPS produced more exosomes（ P〈0.01）.Exosomes from BMSCs with or without LPS treatment could increase the ratio of Ly6C^（high） monocytes（ P〈0.01） and downregulate the ratio of Ly6C^（high） macrophages（ P〈0.05）,and the effect of LPS treated-exosomes was more significant than untreated-exosomes（ P〈0.05）.Moreover,the concentration of IL-6 was also elevated under exosomes treatment（ P〈0.05）.Conclusions Human bone marrow mesenchymal stem cells-derived exosomes contribute to the regulation of Ly6C^（high） monocytes/macrophages,indicating that they could be involved in the therapeutic treatment of inflammatory diseases.

关 键 词：骨髓间充质干细胞 外泌体 脂多糖 Ly6C 单核细胞 巨噬细胞 

分 类 号：R-33[医药卫生] R329.24