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| 旋转超滤：一种提取细胞外泌体的新方法 |
| 胡国文1,李青2,牛鑫2,胡斌2,刘鹃1,沈晓黎1,汪泱2*,邓志锋1,3* |
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(1. 南昌大学第二附属医院神经外科, 南昌 330006;
2. 上海交通大学医学院附属第六人民医院四肢显微外科研究所, 上海 200233;
3. 上海交通大学医学院附属第六人民医院神经外科, 上海 200233
*通信作者) |
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| 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，0.22 μm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100 nm，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。 |
| 关键词: 外泌体 间质干细胞 旋转超滤 透射电子显微镜检查 |
| DOI：10.3724/SP.J.1008.2014.00598 |
| 投稿时间：2013-11-25修订日期：2014-03-11 |
| 基金项目:国家自然科学基金（81272170），江西省创新团队计划（20113BCB24018）. |
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| Stirring ultrafiltration：a new method to isolate exosome |
| HU Guo-wen1,LI Qing2,NIU Xin2,HU Bin2,LIU Juan1,SHEN Xiao-li1,WANG Yang2*,DENG Zhi-feng1,3* |
(1. Department of Neurosurgery, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China;
2. Institute for Microsurgery of Extremities, the Sixth People's Hospital of Shanghai, Shanghai Jiaotong University School of Medicine, Shanghai 200233, China;
3. Department of Neurosurgery, the Sixth People's Hospital of Shanghai, Shanghai Jiaotong University School of Medicine, Shanghai 200233, China
*Corresponding authors.) |
| Abstract: |
| 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. |
| Key words: exosome mesenchymal stem cells stirring ultrafiltration transmission electron microscopy |
