人羊膜间充质干细胞来源外泌体的提取与鉴定

目的探索人羊膜间充质干细胞来源外泌体(hAMSC-Exos)的分离、纯化与鉴定方法,为外泌体的生物学功能研究奠定基础。方法由废弃的羊膜提取间充质干细胞;采取改良超速离心法分离、纯化培养液上清中的hAMSC-Exos;透射电镜观察、纳米粒子跟踪分析技术(NTA)、Western blot和流式细胞术用于hAMSC-Exos的鉴定外泌体。结果成功用改良超速离心法分离出hAMSC-Exos,透射电镜(TEM)下观察呈中间凹陷的茶杯样结构;NTA结果显示样品直径符合外泌体直径大小;Western blot和流式细胞术证实外泌体表面特异性蛋白CD9、CD63、CD81表达阳性。结论建立了hAMSC-Exos的提取纯化及鉴定方法,为它后续的研究提供依据。

The isolation and identification of human amniotic mesenchymal stem cell-derived exosomes

Objective The isolation,purification and identification of exosomes were studied from human amnion mesenchymal stem cells(hAMSCs)to provide a basis for the study of exosomal biological functions.Methods Mesenchymal stem cells were extracted from the discarded amniotic membrane.Exosomes in the culture supernatant were isolated and purified by a modified ultracentrifugation method and identified by TEM,NTA,Western blot and flow cytometry.Results hAMSC-Exos were successfully separated by modified ultracentrifugation,TEM revealed that hAMSC-Exos exhibited a cup-shaped structure with recessed in the middle;NTA detection showed that the diameter of sample was consistent with exosomes;Western blot and Flow cytometry confirmed that the positive expression of the surface-specific surface marker proteins CD9,CD63,and CD81.Conclusion The isolation and identification of hAMSC-Exos has been established successfully,which would provide a basis for the subsequent research of the hAMSC-Exos.