一种绿色荧光蛋白标记的骨髓间充质干细胞源性外泌体的构建及鉴定  

Establishment and identification of the green fluorescence labeled exosomes in rat bone marrow mesenchymal stem cells

作　　者：任瑞[1] 谭晓红[1] 鲁亚成[2] 刘辉[1] 张显芳[1] 付媛[1] 黄奎[1] 黄奕弟[1] 马志健[1] 易西南[1] 

机构地区：[1]海南医学院人体解剖学教研室,海口571101 [2]第四军医大学解剖学教研室暨梁銶琚脑研究中心,西安710032

出　　处：《神经解剖学杂志》2017年第2期167-171,共5页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81460196);海南省自然科学基金创新团队项目(2016CXTD009)

摘　　要：目的:构建稳定表达绿色荧光蛋白(GFP)的骨髓间充质干细胞(bone marrow mesenchymal stem cells,MSCs),获得GFP标记的MSCs源性的外泌体(exosomes)。方法:全基因合成CD63序列,连接到穿梭载体,酶切验证后转染293T细胞,测定病毒滴度后感染MSCs,72 h后用嘌呤霉素筛选稳定表达的细胞。荧光定量PCR检测CD63在MSCs中的表达。取其无血清培养72 h的上清液,获得提取物,经透射电镜和Western Blot验证。结果:提取物为类圆形囊泡,大小约为40~100 nm,具有CD63和Alix表达。结论:成功构建携带GFP标记的MSCs源性的外泌体,为研究外泌体在细胞中的摄取及信号传递提供了工具。Objective：To prepare for the further studies of the intake and signal transduction of exosomes in cells,established rat bone marrow mesenchymal stem cells （MSCs） that produce the exosomes labeled by GFP.Methods：CD63 gene was connected to the shuttle vector,and the recombinant pasmid was transfected into 293T cells,and virus titer tested.The stable expression of MSCs was selected by puromycin after 72 hours infection.The expression of CD63 in MSCs was detected by quantitative fluorescence PCR.Take the serum-free culture supernatant of 72 hours to obtain the extract,and detected it by transmission electron microscopy and Western blot.Results：The extract was a homogeneous and uniform circular vesicle about 40 ～ 100 nm diameter with expression of CD63 and Alix.Conclusion：The exosomes from MSCs labeled by GFP were successfully constructed and identified.

关 键 词：外泌体 骨髓间充质干细胞 绿色荧光蛋白 

分 类 号：R745[医药卫生—神经病学与精神病学]