人脑胶质瘤细胞来源外泌体的提取鉴定及微环境中的摄取

Extraction and identification of exosomes from human glioma cells and uptake in microenvironment

目的探讨提取、鉴定及观察细胞对外泌体在微环境中的摄取,为后续开展胶质瘤外泌体相关的研究建立稳定的提取方法,以及更清楚地观察到外泌体奠定基础,为今后研究提供一定的参考。方法运用超速离心法从人脑胶质瘤细胞培养上清液中分离外泌体以及观察在肿瘤细胞微环境中肿瘤细胞对外泌体的摄取,通过透射电子显微镜观察外泌体的大小、形态特点,通过马尔文粒径分析确定外泌体粒径的大小,应用Western blotting法进行外泌体特异性抗原分子CD63、TSG101的鉴定,采用荧光共聚焦显微镜观察胶质瘤细胞摄取外泌体。结果通过透射电子显微镜观察到从人脑胶质瘤U251细胞的培养上清液中分离出来的呈圆形杯盘样结构的囊泡样物质,其外形呈典型的杯状和双凹圆盘的扁平球囊体且具备完整的膜结构,粒径大小为30~150 nm,Western blotting结果显示其表达外泌体特异性蛋白CD63、TSG101;外泌体通过细胞内吞噬作用形成、分泌和释放,通过荧光共聚焦显微镜可以证实其有效摄取外泌体,主要集中于细胞质。结论人脑胶质瘤U251细胞中提取分离得到具有双层膜结构茶托样囊泡物质,鉴定其为外泌体,并能观察细胞摄取外泌体集中分布于细胞质。

Objective To extract,identify and observe the uptake of cellular exosomes in the microenvironment,so as to estab⁃lish a stable extraction method and observe exosomes more clearly for the subsequent studies on glioma exosomes,and to provide cer⁃tain reference for the current studies on exosomes.Methods Use ultracentrifugation to separate exosomes from the culture superna⁃tant of human glioma cells and observe the uptake of exosomes by tumor cells in the tumor cell microenvironment.Observe the size and morphological characteristics of exosomes through transmission electron microscopy.The particle size of exosomes was determined by Malvern particle size analysis.Western blotting was used to identify the exosomes-specific antigen molecules CD63 and TSG101.Confocal fluorescence microscopy was used to observe the uptake of exosomes by glioma cells.Results The vesicle-like substance isolated from the culture supernatant of human glioma U251 cells was characterized by typical cup-shaped and double hollow disc flat balloon,round saucer-like vesicle and complete membrane structure with particle size of 30-150 nm,and expressed exo⁃somes-specific proteins CD63 and TSG101.Exosomes were formed,secreted and released in the endocytosis system,and the effective uptake of exosomes was confirmed by fluorescence confocal microscopy,which was mainly concentrated in the cytoplasm.Conclu⁃sion Vesicle-like substances were successfully isolated from human glioma U251 cells and identified as exosomes,and cellular up⁃take of exosomes was observed to be concentrated in the cytoplasm.