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山东大学学报（医学版） ›› 2015, Vol. 53 ›› Issue (1): 6-9.doi: 10.6040/j.issn.1671-7554.0.2014.348

• 基础医学 • 上一篇    下一篇

小鼠胰岛微血管内皮细胞外泌体的提取和鉴定方法

肖芳1,2,3, 蒋卫东1, 王晓红1, 秦爱琼1, 韩敏1, 陈丽2,4   

  1. 1. 山东大学第二医院干部保健科, 山东 济南 250033;
    2. 山东大学内分泌代谢病研究所, 山东 济南 250033;
    3. 山东大学细胞治疗研究中心, 山东 济南 250033;
    4. 山东大学齐鲁医院内分泌科, 山东 济南 250012
  • 收稿日期:2014-05-28 修回日期:2014-10-22 发布日期:2015-01-10
  • 通讯作者: 陈丽。E-mail:chenli3@medmail.com.cn E-mail:chenli3@medmail.com.cn
  • 基金资助:
    山东省科技攻关国际科技合作项目(2010GHZ20201);济南市科技局科技明星项目(20100318)

Extraction and identification of mouse islet microvascular endothelial cells (MS-1)-derived exosomes

XIAO Fang1,2,3, JIANG Weidong1, WANG Xiaohong1, QIN Aiqiong1, HAN Min1, CHEN Li2,4   

  1. 1. Department of Cadre Health Care, Second Hospital of Shandong University, Jinan 250033, Shandong, China;
    2. Institute of Endocrinology and Metabolism, Shandong University, Jinan 250033, Shandong, China;
    3. Research Centre for Cell Therapy, Shandong University, Jinan 250033, Shandong, China;
    4. Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China
  • Received:2014-05-28 Revised:2014-10-22 Published:2015-01-10

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摘要： 目的 建立小鼠胰岛微血管内皮细胞来源的外泌体的提取和鉴定方法,为糖尿病胰岛微血管内皮受损的发病机制研究及重建胰岛微循环完整性的应用提供必要材料。方法 选取小鼠胰岛微血管内皮细胞株(MS-1)细胞作为研究对象, 取其培养上清液, 经多步离心结合蔗糖/D2O垫超速离心方法获取提取物,然后采用透射电镜和Western blotting 法对提取物进行形态和蛋白成分分析,以确定其是否为外泌体。结果 提取物呈形态均一的类圆形囊泡,有完整双层膜包绕,内部含有低电子密度物质。囊泡大小约为40～100 nm,可散在分布,也可聚集成团,提取物均阳性表达CD63、CD9和TSG101分子。结论 经过形态和蛋白成分分析证实所提取到的物质正是MS-1细胞来源的外泌体,便于后续临床和基础研究工作的开展。

关键词: 小鼠胰岛内皮细胞, 提取, 离心, 外泌体

Abstract: Objective To establish a method for mouse islet microvascular endothelial cells (MS-1)-derived exosome extraction and identification. Methods Exosomes from the supernatant of MS-1 cells were collected by the combination of step-by-step centrifugations and ultracentrifugation, then characterized by transmission electron microscopy (TEM) and Western blotting. Results The extract was of round vesicle structure, surrounded by an intact double membrane, containing low electron density material inside. The diameters of exosomes ranged from 40-100 nm. The exosomes scattered or gathered into a micelles, and positively expressed CD63, CD9, and TSG101 molecules. Conclusion The extracts are proved to be exosomes and the method for extraction and identification will facilitate further clinical and basic researches.

Key words: Mouse islet endothelial cells, Exosome, Centrifugation, Isolation

中图分类号: 

  • R587.1
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