摘要
目的建立一种利用旋转超滤技术从骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)培养上清液中分离外泌体(exosome)的方法。方法收集BMSCs细胞培养上清,低速离心去除残余细胞,0.22μm滤器过滤除去细胞碎片,采用旋转超滤技术提取外泌体。应用透射电子显微镜观察所获外泌体的形态,用蛋白质印迹法检测外泌体标记物CD63、CD9蛋白的表达,用CCK-8试剂盒检测外泌体对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖活性的影响。结果使用旋转超滤技术成功分离出BMSCs分泌的外泌体,透射电镜下外泌体为圆形或椭圆形,直径约100nm,蛋白质免疫印迹法检测结果显示外泌体中有其特异标记物CD63、CD9蛋白的表达。外泌体能够促进HUVECs增殖,并且其促细胞增殖作用随浓度增高而增强。结论旋转超滤技术是一种简单有效的提取外泌体的方法。
Objective To establish a method using stirring ultrafiltration for isolating exosome from the culture supernatant of bone marrow mesenchymal stem cells（BMSCs）. Methods The culture supernatant of BMSCs was collected and centrifuged at low speed to remove residual cells, and then the cell debris was removed by 0. 22 μm filter. Then the clarified supernatant was purified by stirring ultrafiltration to obtain exosome. Transmission electron microscope was used to identify the morphology of exosome, Western blotting analysis was used to examine CD63, CD9 protein expression, and CCK-8 kit was employed to detect the effect of exosome on proliferation of human umbilical vein endothelial cells（HUVECs）. Results Stirring ultrafiltration successfully isolated exosome from BMSCs culture supernatant; morphology of the isolated exosome was round or elliptic, with the diameter being about 100 nm. Results of Western blotting analysis showed positive expression of CD63, CD9 proteins. The exosome promoted proliferation of HUVECs in a dose-dependent manner. Conclusion Stirring ultrafiltration technique is a simple and efficient method for exosome isolation.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2014年第6期598-602,共5页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(81272170)
江西省创新团队计划(20113BCB24018)~~
关键词
外泌体
间质干细胞
旋转超滤
透射电子显微镜检查
exosome
mesenchymal stem cells
stirring ultrafiltration
transmission electron microscopy
