摘要
目的建立稳定、高效的骨髓间充质干细胞(BM-MSCs)培养及外泌体提取方法。方法通过全骨髓、差速贴壁并辅助以低氧(5%O2)环境的方法进行小鼠BM-MSCs培养,收集细胞,鉴定干细胞表面标志物,进行成骨、成脂及成软骨诱导;并收集上清液,超速分离的方法提取外泌体后鉴定。结果小鼠BM-MSCs生长状态好,第3代细胞就可达到95%以上的纯度且可稳定传代至15代左右,表达间充质干细胞(MSCs)标志物,且可被成功诱导分化成骨、成脂肪及软骨组织。超速离心得到的细胞外囊泡具有典型外泌体样结构,Western blot鉴定显示具有外泌体特征性蛋白标志物。结论全骨髓培养方法使得小鼠BM-MSCs的损失最少,低氧(5%O2)环境下的差速贴壁法使得小鼠BM-MSCs增殖速度快,纯度高。该方法培养的小鼠BM-MSCs可稳定分泌外泌体囊泡,为小鼠MSCs的培养及其外泌体获取提供了良好的方法。
Objective To establish a stable and efficient method for the culture of mouse bone marrow mesenchymal stem cells and the extraction of exosomes. Methods The whole bone marrow, differential attachment and hypoxia environment were used to culture bone marrow mesenchymal stem cells(BM-MSCs).Then the cells were collected, the surface markers of stem cells were identified, and osteogenesis, adipogenesis and chondrogenesis were induced. The supernatant was collected, and the exosomes were extracted by overspeed separation and identified. Result The growth of mouse BMSCs was great, and the purity of the third generation cells could reach more than 95% and stable passage to about 15 generations. The cells expressed the markers of mesenchymal stem cells, and could be successfully induced to differentiate into osteogenesis,adipogenesis and chondrogenesis. The extracellular vesicles obtained by ultracentrifugation had typical exosome-like structure, and Western blot identification showed that they had characteristic protein markers of exosomes. Conclusion The whole bone marrow culture method makes the loss of BMSCs in mice minimum, and the differential attachment method under 5% oxygen environment makes the proliferation of BMSCs in mice fast and high purity. Moreover, the mouse BMSCs cultured by this method can secrete exosomes vesicles steadily. This provides a great method for the culture of mouse mesenchymal stem cells and the acquisition of their exosomes.
作者
程欢
苏盖
陶思跃
尤涛
Cheng Huan;Su Gai;Tao Siyue(Dept of Orthofaedics,The First Affiliated Hospital qf Anhui Merical University,Hefei 230022;Dept of Orthofaedics,Huosian County Hospital,Lu'an 237200)
出处
《安徽医科大学学报》
CAS
北大核心
2020年第4期518-522,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81171797)。
关键词
小鼠
骨髓间充质干细胞
全骨髓
差速贴壁
低氧培养
外泌体
mouse
bone marrow mesenchymal stem cells
whole bone marrow
differential attachment
hypoxic culture
exosomes
