小鼠胰岛微血管内皮细胞外泌体的提取和鉴定方法

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

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。

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 combina-tion 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 fur-ther clinical and basic researches.