人脐带间充质干细胞来源的微囊泡提取和鉴定

Isolation and identification of human umbilical cord mesenchymal stem cells-derived microvesicles

目的研究分离和提纯人脐带间充质干细胞来源的微囊泡(hUC-MSCs-MVs)并分析其理化性质。方法 (1)运用Ⅰ型胶原酶联合透明质酸酶消化Wharton's Jelly组织的方法,分离和扩增人脐带间充质干细胞(hUC-MSCs);(2)实验组:用"饥饿法"处理hUC-MSCs,分离和提纯hUC-MSCs-MVs;对照组:用未经"饥饿法"处理的hUC-MSCs来尝试提取hUCMSCs-MVs;(3)验证hUC-MSCs-MVs的理化性质和生物学特性。结果 (1)分离出的hUCMSCs状态良好,扩增后的子代hUC-MSCs数量及状态满足后续实验的要求。流式细胞术结果显示hUC-MSCs-MVs高表达CD44、CD29和CD73,而低表达α-6 integrin(CD49f)和HLAclassⅡ(HLA-DR)。(2)实验组成功分离和提纯了hUC-MSCs-MVs,而对照组未见确切hUCMSCs-MVs产生;扫描电镜观察到hUC-MSCs-MVs的囊泡样结构,直径几十到几百nm不等,均在1μm以下;透射电镜观察到hUC-MSCs-MVs的产生和胞吐作用;蛋白定量结果为1337.1μg/ml;流式细胞术结果显示其大小绝大部分在1μm以下,并能高表达CD44和CD29,而低表达CD73、α-6 integrin(CD49f)和HLA-classⅡ(HLA-DR)等表面分子。结论用"饥饿法"处理hUC-MSCs可成功获得hUC-MSCs-MVs。

Objective The purpose of this present study was to isolate, purify human umbilical cord mesenchymal stem cells （hUC-MSCs） derived microvesicles （hUC-MSCs- MVs）, and to analyze the physicochemical properties and biological characteristics of them. Methods （1）HUC-MSCs were isolated by the Wharton＇s Jelly by type I collagenase combined with hyaluronidase digestion, and then they were further cultured in DMEM medium containing 10 % FBS. （2）Experimental group： hUC-MSCs-MVs were isolated and purified from hUC-MSCs that were processed with ＂starvation method＂. Control group： normal supernatant of hUC-MSCs were processed to isolate and purify hUC-MSCs-MVs. （3）The physicochemical properties and biological characteristics of hUC-MSCs-MVs were verified by flow cytometry （FCM） and electron microscope. Results （1）FCM showed hUC-MSCs expressed CD44, CD29 and CD73. Expression of α-6 integrin （CD49f） and HLA-class Ⅱ （HLA-DR） was low. （2）The isolation and purification of hUC-MSCs- MVs were successful in the experimental group, while no hUC-MSCs-MVs were isolated in the control group. The sizes of hUC-MSCs-MVs measured by SEM were generally below 1 μm, and the formation and exocytosis of hUC-MSCs-MVs were observed in hUC-MSCs by TEM. The protein concentration of hUC-MSCs-MVs was about 1337.1 μg/ml. The results of the FCM showed that the diameter of hUC-MSCs-MVs were generally below 1 μm, with high expression of CD44 and CD29 but low expression of CD73, α-6 integrin （CD49f） and HLA-class Ⅱ（HLA-DR）. Conclusions The hUC-MSC-MVs can be successfully isolated and purified from hUC-MSCs by the ＂starvation method＂.