摘要
目的:探索来源于人脐带间充质干细胞(hUC-MSCs)的工程化纳米囊泡(Nanovesicle,NVs)的制备与鉴定方法,为深入了解工程化NVs的生物学功能提供数据基础。方法:采用物理方式破碎hUC-MSCs以获取NVs,并采用改良的超速离心法进行分离和纯化;通过纳米颗粒跟踪分析(NTA)、透射电镜(TEM)观察、蛋白质免疫印迹(WB)及生物学活性分析方法鉴定NVs的特性。结果:建立的方法成功分离出NVs,TEM下观察到NVs呈中间凹陷的茶杯样结构;NTA结果显示NVs直径在100 nm左右;Western Blot实验证实NVs几乎不表达外泌体特异性蛋白TSG101;生物学活性实验表明NVs不能促进人皮肤成纤维(HSF)细胞增殖。结论:本研究建立的hUC-MSCs来源的NVs提取及鉴定方法可行,为NVs在未来的临床研究提供了数据基础。
Objective:The purpose of this present study is to explore the preparation and identification methods of engineered nanovesicles(NVs)derived from human umbilical cord mesenchymal stem cells(hUC-MSCs),and to provide a data foundation for further understanding of the biological functions of engineered NVs.Methods:Physical methods are employed to break down hUC-MSCs in order to obtain NVs,which are then separated and purified using a modified ultracentrifugation method.The characteristics of NVs are identified using methods including nanoparticle tracking analysis(NTA),transmission electron microscopy(TEM),Western Blot(WB),and biological activity analysis.Results:Using our established method,NVs are successfully prepared,which show a cup-shaped structure with a concave middle when observed under TEM;NTA results demonstrate that the diameter of NVs is approximately 100 nm;Western Blot analysis confirmed the extremely low expression of the exosome-specific protein TSG101 in NVs,and functional experiments demonstrated that NVs could not promote proliferation of human skin fibroblasts(HSF).Conclusion:The preparation and identification of NVs derived from hUC-MSCs has been established successfully,which provides a data foundation for future clinical research on NVs.
作者
李磊
杜飞跃
杨文杰
LI Lei;DU Feiyue;YANG Wenjie(Nanjing Jinyang Biotechnology Co.,Ltd.,Nanjing 211899,China)
出处
《生物化工》
CAS
2023年第5期143-146,151,共5页
Biological Chemical Engineering
关键词
人脐带间充质干细胞
外泌体
纳米囊泡
制备与鉴定
human umbilical cord mesenchymal stem cells
extracellular vesicles
nanovesicles
preparation and identification
