小鼠骨髓间充质干细胞外泌体的分离与鉴定

Isolation and identification of exosomes derived from bone marrow mesenchymal stem cells

目的探讨骨髓间充质干细胞的外泌体的分离与鉴定方法。方法通过全骨髓贴壁法培养间充质干细胞;用新兴试剂法收集外泌体,采用电镜、粒径检测、流式细胞仪和Western blot鉴定外泌体。结果第三代骨髓间充质干细胞表面CD45呈阴性表达,CD73和CD105呈阳性表达;骨髓间充质干细胞外泌体呈圆形或椭圆形,大小不均匀,直径30~100 nm,有完整的膜结构;粒径检测显示颗粒直径主峰为61.25 nm,直径为20~200 nm的颗粒占72.4%;外泌体CD63和CD81呈阳性表达;细胞培养上清液中可检测到外泌体CD9和CD63的蛋白表达。结论在培养间充质干细胞的培养基中可以收集到外泌体,可以通过透射电镜、粒径检测、流式细胞仪和Western blot对骨髓间充质干细胞的外泌体进行鉴定。

Objective To develop a method of isolation and identification of exosomes of bone marrow mesenchymal stem cells. Methods The mesenchymal stem cells were cultured by whole bone marrow adherence method. A new reagent-exosomes extraction kit was used to isolate and collecte exosomes. The exosomes were identified by electron microscopy,particle size detection,flow cytometry and Western blot.Results The expression of CD45 on the surface of the third generation bone marrow mesenchymal stem cells was negative,and CD73 and CD105 were positive; exosomes derived from bone marrow mesenchymal stem cells were round or oval,the size is non-uniform,the diameter is 30 ～ 100 nm,have a complete membrane structure,and containing low-density substances; Particle size detection particle diameter of the main peak was 61. 25 nm,in which the diameter of particles was about 20-200 nm accounted for 72. 4%; exosome expressed CD63 and CD81; The expression of CD9 and CD63 from cell culture supernatants was positive. Conclusions The exosomes can be collected in the medium of mesenchymal stem cells. The exosomes derived from bone marrow mesenchymal stem cells can be identified by electron microscopy,particle size detection,flow cytometry and Western blot.