大鼠骨髓间充质干细胞来源的胞外囊泡分离和鉴定  

Isolation and identification of extracellular vesicles from rat bone marrow mesenchymal stem cells

作　　者：葛锁华[1] 马永宾[1] 芮星[1] 张俊华[1] 汪雪峰[2] 苏建华[1] GE Suohua;MA Yongbin;RUI Xing;ZHANG Junhua;WANG Xuefeng;SU Jianhua(Jintan Hospital, Jiangsu University, Jintan 213200;Hospital Affiliated to Jiangsu University, Zhenjiang 212001, China)

机构地区：[1]江苏大学附属金坛医院,江苏金坛213200 [2]江苏大学附属医院,江苏镇江212001

出　　处：《细胞与分子免疫学杂志》2017年第10期1381-1384,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：江苏省社会发展项目(BE2017697);江苏省"六大人才高峰"项目(WSN-009);江苏省自然科学基金(BK20141295);江苏省"333工程"项目(BRA2014172);镇江市社会发展项目(SH2015033);镇江市重点医学人才(2014);江苏大学附属医院科技项目(jdfyRC2015010);江苏省"六个一"工程(LGY2016055);江苏大学医学临床科技发展基金(JLY20160081)

摘　　要：目的探讨大鼠骨髓间充质干细胞来源的胞外囊泡(BMSC-EV)的分离、鉴定及-80℃冻存对BMSC-EV膜结构完整性的影响。方法应用超高速离心法从大鼠BMSC培养上清液(BMSC-CS)中分离并纯化EV,扫描电镜和透射电镜观察其大小和形态;Western blot法检测EV标志性蛋白CD63的表达;-80℃冻存后,不同时间段通过透射电镜观察EV膜结构的完整性。结果扫描电镜观察BMSC表面附着有大量膜结构的EV,形态呈圆形或椭圆形,直径约50~1000 nm;透射电镜观察BMSC-EV具有膜结构,形态和大小与扫描电镜结果一致;Western blot结果表明,BMSC-EV表达CD63;-80℃冻存后,在第1个月和第3个月分别通过透射电镜观察BMSC-EV膜结构完整,而冻存第6个月BMSC-EV膜结构破裂。结论成功提取了BMSC-EV,且随着冻存时间的延长,其膜结构完整性降低。Objective To investigate the isolation and identification of extracellular vesicles（EVs） from rat bone marrow mesenchymal stem cells（BMSCs） and the effect of-80℃ cryopreservation on the structural integrity of the EVs＇ membrane.Methods EVs were isolated and purified from the culture supernatant of bone marrow mesenchymal stem cells（ BMSC-CS）by ultrahigh velocity centrifugation. The size and morphology of EVs were observed by scanning electron microscopy（ SEM）and transmission electron microscopy（ TEM）. Western blotting was performed to detect the expression of CD63 protein. The integrity of the EVs＇ membrane structure was observed by TEM at different time after storage at-80℃. Results SEM showed that a large number of EVs with membrane structures,round or oval and ranged in size from 50 to 1000 nm were attached onto BMSC surface. Under TEM,BMSC-EVs were similar in morphology and size to them under SEM. Western blotting revealed that BMSC-EV expressed the marker protein CD63. BMSC-EVs had a complete membrane structure at the first month and the third month,whereas BMSC-EV＇s membrane structure ruptured at the sixth month after storage at-80℃. Conclusion BMSC-EVs is successfully extracted, and the membrane structure integrity get worse with the prolongation of cryopreservation time.

关 键 词：骨髓间充质干细胞 胞外囊泡 分离 鉴定 

分 类 号：R392-33[医药卫生—免疫学] Q345.22[医药卫生—基础医学]