人骨髓间充质干细胞体外定向诱导分化为心肌样细胞的超微结构

Ultrastructure of cardiomyocytes differentiated from human marrow mesenchymal stem cells in vitro

目的:培养人骨髓间充质干细胞(hMSCs),体外向心肌细胞(CMs)定向诱导分化,进行超微结构观察,为hMSCs体外向CMs诱导分化提供理论依据。方法:体外分离培养扩增hMSCs并进行流式细胞仪分析鉴定;选用生长良好,纯度高的P5代hMSCs,用5-氮杂胞苷(5-Aza,10μmol.L-1)体外定向向CMs诱导分化,对其进行Desmin检测,在倒置显微镜及透射电镜下观察细胞形态学和超微结构。结果:流式细胞仪检测显示体外分离纯化培养扩增出细胞不表达CD34,高表达CD44,表明扩增细胞为hMSCs。hMSCs经5-Aza诱导分化后其形态发生改变,逐渐由长梭形变为多角形及星形,并表达Desmin。透射电镜下可见细胞大小不一致,细胞表面有许多微绒毛;细胞器丰富,胞质内可见丰富的线粒体、粗面内质网;部分细胞内可见大量糖原颗粒沉积,小部分细胞内可见髓样小体;胞浆内可见肌丝样结构,细胞间存在细胞连接。结论:体外分离培养扩增的hMSCs经5-Aza诱导,可分化成具有CMs超微结构特性的心肌样细胞。

Objective To culture human mesenchymal stem cells （hMSCs） and study the uhrastructure of their myogenic differentiated cells in vitro, and provid the theoretical foundation for hMSCs differentiation into cardiomyocytes （CMs）. Methods hMSCs were cultivated and expanded by adhereing peculiarity. The phenotypes of hMSCs were identified by fluorescent - activated cell sorter （FACS）. With good growth characteristics and high purity, the fifth passage hMSCs were treated for 24 h with 5-azacytidine （5-Aza, 10 μmol · L^-1 ） to induce cellular differentiation. The expression of desmin, identified by immunohistochemistry, was used to identify cardiac muscle differentiation. The morphology and uhrastructure of treated hMSCs were observed by inverted microscope and transmission electron microscope. Results The cultured cells did not express CD34 and expressed a high level of CD44 by FACS, which indicated that they were hMSCs. The morphology of hMSCs treated with 5-Aza under inverted microscope changed from fusiform to polygon or astrocyte, and the induced hMSCs demonstrated positive staining for desmin. With transmission electron microscope, the uhrastructure of the treated hMSCs was visible.The magnitude of cells was not identical, the surface of some ceils had abundance of microvilli. Intracytoplasm had rich organelles, mitochondrium and rough endoplasmic reticulum. In the part of ceils, the sedimentary hepalin granules were observed and only the myeloid body could be seen in small part of cells. Myofilaments were found in cytoplasm, and between cells there were celt iunctions. Conclusion After treated with 5-Aza, hMSCs cultivated and expanded in vitro could differentiate into cardiomyocyte-like cells with CMs＇ uhraslructure cbaracteristics.