人骨髓间质干细胞来源外体的分离鉴定及生物学特性

Extraction and identification for exosome of mesenchymal stem cells derived from human bone marrow and its biological characteristics

目的分离纯化人骨髓间质干细胞(MSCs)来源的外体(exosome),并鉴定其生物学特性。方法收集人骨髓MSCs培养上清,采用超滤及梯度离心法分离并纯化exosome,通过透射电镜观察其形态,SDS-PAGE电泳初步分析exosome蛋白质组成,westernblot检测exosome包含的相关蛋白质,用流式细胞术检测其表面标志CD44、CD29等。结果骨髓来源的MSCs能够分泌exosome,电镜下呈椭圆或碟状的囊泡结构,直径约40～100nm。exosome内含多种蛋白质成分,具有MSCs膜脂质成分,能与PKH26脂质染料结合,并表达MSCs表面黏附分子CD44、CD29。结论人骨髓MSCs能够分泌exosome,采用超滤及梯度离心法可以成功从MSCs培养上清中分离纯化exosome,MSCs分泌的exosome具有MSCs的胞膜及胞质蛋白组分。本研究可为进一步开展人骨髓MSCs来源exosome的生物学特性及其在组织损伤修复中的作用和机制研究提供实验基础。

Objective To extract and purify exosome of mesenchymal stem cells （MSCs） derived from human bone marrow and identify its biological characteristics.Methods The cell culture supernatant of MSCs from human bone marrow was collected.Exosome was extracted and purified with ultrafiltration and gradient centrifugation.The morphology of exosome was observed by transmission electronic microscope and the protein composition of exosome was initially analyzed by SDS-PAGE.The associated proteins of exosome were identified by western blot and the expressions of CD44 and CD29 in exosome of MSCs were analyzed by fluorescent activated cell sorter （FACS） .Results The MSCs from human bone marrow secreted exosome which exhibited elliptic or saucer-like shape and its diameter ranged from 40 to 100 nm with membrane structure.The protein of exosome contained diverse components,e.g.,plasmalemmal and cytoplasmic proteins of MSCs from human bone marrow.Exosome can be labeled by PKH26 and express the surface adhesion molecules CD44 and CD9 of MSCs.Conclusions Exosome may be secreted by MSCs of human bone marrow and could be extracted and purified by ultrafiltration and gradient centrifugation.The exosome secreted by MSCs contains plasmalemmal and cytoplasmic proteins of bone marrow MSCs.This study may provide experiment basis for the further research on the biological characteristics and function of exosome from MSCs and the mechanism of exosome in the repair of tissue damage.