人脐带间充质干细胞来源外泌体的提取、鉴定和蛋白组学分析  

Extraction,identification and proteomic analysis of exosomes derived from human umbilical cord mesenchymal stem cells

作　　者：单政铭 陶述春 胡春梅 张治元 丁一楠 何梦铖 唐秋莎[1] Shan Zhengming;Tao Shuchun;Hu Chunmei;Zhang Zhiyuan;Ding Yinan;He Mengcheng;Tang Qiusha(Department of Pathology and Pathophysiology,School of Medicine,Southeast University,Nanjing 210000,Jiangsu Province,China)

机构地区：[1]东南大学医学院病理与病理生理学系,江苏省南京市210000

出　　处：《中国组织工程研究》2022年第19期3036-3042,共7页Chinese Journal of Tissue Engineering Research

基　　金：江苏社会发展项目(BE2018606),项目负责人:胡春梅;江苏省六大人才高峰(wsw-028),项目负责人:张治元;南京市卫生科技发展专项资金项目计划(ZKX18042),项目负责人:胡春梅。

摘　　要：背景:现有的外泌体蛋白组学研究中,只有不同来源外泌体或者不同条件下分泌的外泌体之间的蛋白组学对比,没有外泌体和其母体细胞之间的对比分析。目的:对人脐带间充质干细胞来源外泌体进行提取鉴定和蛋白组学分析。方法:培养人脐带间充质干细胞并采用超滤序贯超离法提取外泌体,通过透射电镜、纳米颗粒跟踪分析、蛋白质定量、蛋白质印迹法及蛋白组学分析等技术鉴定。结果与结论:(1)提取的外泌体分散度较好且均一,多为杯状圆形或类圆形的膜性小囊泡,可见囊泡的双膜性结构,中央为低电子密度成分,分布较集中且边界清晰;(2)外泌体粒径分布峰值为(129.5±8.7)nm,膜表面带负电荷,浓度为8.375×10^(10)粒子/mL,平均zeta电位为(-28.1±3.6)mV;(3)外泌体有特征性膜蛋白CD9、CD63的表达;(4)蛋白组学分析外泌体中高表达的蛋白质大多参与RNA剪接、mRNA加工、蛋白折叠等生物过程,参与RNA/DNA等遗传物质及蛋白质的合成、加工、降解过程,参与组成多种细胞器及亚细胞膜结构,参与多条信号通路、细胞黏附、细胞外基质受体相互作用,与多种疾病和病毒致癌也密切相关;(5)通过分析认为外泌体与其母体细胞相比有一定的同质性和差异性,差异蛋白功能均和免疫无关,故验证了人脐带间充质干细胞来源外泌体的低免疫原性和安全性。BACKGROUND:Current proteomics studies of exosomes only have proteomic comparisons between exosomes from different sources or exosomes secreted under different conditions.There is no comparative analysis between exosomes and their parent cells.OBJECTIVE:To extract,identify and analyze the exosomes of human umbilical cord mesenchymal stem cells.METHODS:Human umbilical cord mesenchymal stem cells were cultured and exosomes were extracted using sequential ultrafiltration and identified using transmission electron microscope,nanoparticle tracking analysis,bicinchoninic acid,western blot assay,and proteomics analysis.RESULTS AND CONCLUSION:(1)The extracted exosomes had good dispersion and uniformity.Most of them were cup-shaped or round-like membranous vesicles.The double membranous structure of the vesicles could be seen,with low electron density components in the center,which were more concentrated and distributed.The boundary was clear.(2)The peak size distribution of exosomes was(129.5±8.7)nm.The membrane surface was negatively charged;the concentration was 8.375×10^(10) particles/mL;and the average zeta potential was(-28.1±3.6)mV.(3)Exosomes showed the expression of characteristic membrane proteins CD9 and CD63.(4)Proteomics analysis showed that most of the highly expressed proteins in exosomes were involved in biological processes,such as RNA splicing,mRNA processing,and protein folding,involved in the synthesis,processing,and degradation of RNA/DNA and other geneticmaterial and proteins,and participated in the composition of organelles and subcellular membrane structures,involved in multiple signal pathways,cell adhesion,extracellular matrix receptor interactions,and were also closely related to a variety of diseases and viral carcinogenesis.(5)It is concluded that after proteomics analysis,it is believed that the exosomes have certain homogeneity and differences compared with their parent cells.Differential protein function has nothing to do with immunity.Therefore,the low immunogenicity and safety of exosom

关 键 词：干细胞 人脐带间充质干细胞 外泌体 蛋白组学 免疫原性 安全性 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]