人颌骨间充质干细胞的外泌体在巨噬细胞调控中的作用

目的:探讨颌骨骨髓间充质干细胞分泌的外泌体(OMMSCs-Exo)的特性及其在调控巨噬细胞功能中可能发挥的作用.方法:体外培养OMMSCs,检测其克隆形成及多向分化能力.提取上清液中外泌体,透射电镜观察其形态,蛋白印迹法检测其表面特异标志CD63、CD81.将外泌体与巨噬细胞作用24 h后用共聚焦显微镜观察两者的作用方式,实时定量PCR检测外泌体内免疫相关miR-223和miR-let-7c表达及与共培养后巨噬细胞中miRNA表达.结果:人OMMSCs符合间充质干细胞特性,OMMSCs-Exo分泌的外泌体近似圆形,直径在60~160 nm 之间,表达表面标志CD63、CD81:内含有与巨噬细胞调控相关的miRNA 如miR-223 和miR-let-7c 等,能被巨噬细胞吞噬,共培养后巨噬细胞中miR-223含量增加.结论:人OMMSCs-Exo及其携带的miRNA可能参与了巨噬细胞功能的调控.

Exosomes serected by mesenchymal stem cells derived form orofacial bone regulate the function of macrophage

Objective:To investigate the trait of exosomes serected by mesenchymal stem cells derived form orofacial bone(OMMSCs-Exo) and the communication between the exosomes and macrophages.Methods:OMMSCs were isolated from orthognathic surgical sites and cultured by limited dilution.Their cell surface markers were characterized by flow cytometry.the rate of colony formation and the differentiation potential of OMMSCs were evaluated.Exosomes were prepaired from the culture supernatants of OMMSCs(P4-P6).Transmission electron microscopy(TEM) and western blot were used to identify the exosomes.The expression of miRNAs associated with immunity such as miR-223 and miR-let-7c were determined by Real-time RT-PCR.Human peripheral blood mononuclear cells(PBMCs) were isolated from health donor and co-cultured with OMMSCs-Exo.After co-cultured for 24 h,the communication between exosomes and macrophages was tested using a confocal microscope.Results:Human OMMSCs were proved to have the characteristics of mesenchymal stem cells.The diameter of OMMSCs-Exo ranged from 40 to 160 nm.The OMMSCs-Exo expressed CD63 and CD81 and contained miRNAs associated with immune regulation such as miR223 and miR-let-7c.OMMSCs-Exo could be uptaken by macrophages.After co-culture of OMMSCs-Exo with marcrophages for 72 h,miR223 expression in macrophages increased.Conclusion:OMMSCs-Exo has the potential of immune regulation.