人脂肪干细胞及其外泌体的分离与鉴定  

Isolation and identification of human adipose-derived stem cells and its exosomes

作　　者：李洪超 金银鹏[2] 王皙 李莉[2] 王晓今[2] 周荣[2] 陈成伟[2] 傅青春[2] 程明亮 Li Hong-chao;Jin Yin-peng;Wang Xi;Li Li;Wang Xiao-jin;Zhou Rong;Chen Cheng-wei;Fu Qing-chun;Cheng Ming-liang(School of Clinical Medicine, Guizhou Medical University, Guiyang 550004, Guizhou Province, China;the 85th Hospital of PLA, Shanghai Research Center for Hepathopathy, Shanghai 200235, China;Department of Infection, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China)

机构地区：[1]贵州医科大学临床医学院,贵州省贵阳市550004 [2]解放军第八五医院上海肝病研究中心,上海市200235 [3]贵州医科大学附属医院感染科,贵州省贵阳市550004

出　　处：《中国组织工程研究》2018年第13期2033-2038,共6页Chinese Journal of Tissue Engineering Research

基　　金：南京军区医学创新重大课题(14ZX01);中国肝炎防治基金会-天晴肝病研究基金资助课题(TQGB20150104)~~

摘　　要：背景:间充质干细胞如今在科研领域被广泛地研究和应用,许多研究认为其发挥作用的机制很大程度上依赖于其旁分泌的外泌体。目的:分离纯化人脂肪干细胞来源的外泌体,并鉴定其生物学特性。方法:采用胶原酶消化法获得人脂肪干细胞,进行细胞表面分子标志和成骨、成脂分化能力鉴定。运用超滤法从人脂肪干细胞条件培养基中提取外泌体,应用扫描电子显微镜、粒度仪观察所获外泌体的形态和大小,采用抗体芯片检测外泌体所含蛋白质表达。结果与结论:(1)人脂肪干细胞呈梭形、漩涡状生长,细胞表面表达CD73、CD44、CD90、CD105分子,具备成脂、成骨等多向分化潜能,可证实为人脂肪干细胞;(2)大多数外泌体直径均在30-150 nm范围内;扫描电镜显示外泌体呈均一大小的圆杯形态;(3)抗体芯片显示外泌体含FLOT1、ICAM、ALIX、CD81、CD63、ANXA5、TSG101等多种特殊蛋白;(4)以上结果表明实验成功分离得到人脂肪干细胞外泌体。BACKGROUND： Currently, mesenchymal stem cells have been widely explored and applied in scientific research field, and many studies suggest that the underlying mechanism of mesenchymal stem cells mainly relies on its exosomes. OBJECTIVE： To isolate and identify human adipose-derived stem cells and its exosomes, and to identify their biological characteristics. METHODS： Human adipose tissue was digested with collagenase l, and adipose-derived stem cells were isolated and purified. Immunophenotype, osteogenic and adipogenic abilities of adipose-derived stem cells were identified. Exosomes were isolated by using ultrafiltration method. Morphology of exosomes was observed by Nanosight and electron microscope. Expression of proteins in exosomes was detected by antibody array method. RESULTS AND CONCLUSION： Adipose-derived stem cells exhibited long spindle-like or fibroblast-like appearance, expressed CD73, CD44, CD90, CD105 and had the potential to differentiate into many tissues, including bone and adipose tissues. The exosomes had the similar size, with the diameter of 30-150 nm. They possessed many proteins including FLOT1, ICAM, ALIX, CD81, CD63, ANXA5, TSG101, and so on. Findings from the present study indicate the successful isolation of exosomes from human adipose-derived stem cells.

关 键 词：外泌体 干细胞 间充质干细胞 脂肪干细胞 

分 类 号：R394.2[医药卫生—医学遗传学]