人脐带间充质干细胞分离培养及成骨分化 原子力显微镜观察细胞膜表面的超微结构变化

背景:目前,人们对间充质干细胞的观察研究多采用扫描电镜、透射电镜和倒置显微镜等,但是这几种方法对观察细胞膜超微结构及细胞骨架的研究则有不足之处.目的:探讨并优化人脐带间充质干细胞体外获取及培养增殖的方法并鉴定;应用原子力显微镜观察其向成骨诱导过程中超微结构的变化.方法:用酶消化法分离培养人脐带间充质干细胞,通过传代培养,扩增.体外向骨方向诱导分化,并通过碱性磷酸酶钙钴法染色、茜素红染色鉴定其分化能力.流式细胞仪检测人脐带间充质干细胞免疫表型;诱导过程中利用原子力显微镜的高空间分辨率从可视化的角度观察其在诱导前后细胞表面超微结构的变化.结果与结论:采用酶消化法能有效分离纯化人脐带间充质干细胞.接种24 h,贴壁细胞形态多为长梭形,多边形或成纤维细胞样形态,大小均一.流式细胞仪分析第3代细胞均表达CD29、CD44 、CD105,不表达CD34、CD45和HLA-DR.经成骨诱导分化4周后,碱性磷酸酶染色呈强阳性,茜素红染色可见明显钙结节;通过原子力显微镜对分化前后的细胞骨架分析:未分化的干细胞其细胞骨架的微管突出不明显,分化后的细胞骨架其微管明显突出,并呈平行分布状态.

Isolation, culture and osteogenic differentiation of mesenchymal stem cells from human umbilical cord:Ultrastructure of cell membrane observed using atomic force microscope

BACKGROUND: Mesenchymal stem cells (MSCs) are commonly observed under the scanning electron microscope, transmission electron microscope and inverted microscope. However, above-mentioned observation methods have disadvantages on observing ultrastructure of cell membrane and cytoskeleton. OBJECTIVE: To establish a more effective and appropriate method to isolate, culture and identification of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro and to study the ultrastructure of osteogenic differen...