大鼠骨髓间充质干细胞的体外分离与培养

Isolation, purification and identification of rat bone marrow mesenchymal stem cells in vitro

目的 建立体外分离纯化及培养扩增大鼠骨髓间充质干细胞的方法.方法 应用全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞,并进行细胞传代培养.倒置显微镜下观察细胞形态及生长特征,测定细胞生长曲线,通过流式细胞仪检测细胞表面标志物,取第3代细胞分别加入成骨、成脂诱导剂并采用碱性磷酸酶染色及Von Kossa染色鉴定成骨能力,以油红O染色鉴定成脂能力.结果 获取的大鼠骨髓间充质干细胞形态呈均一成纤维细胞样,并呈集落样生长.传至第3代细胞纯度可达97%以上,其细胞表型CD29、CD44、CD105、CD166呈阳性表达,CD34、CD80、CD86呈阴性表达.经成骨诱导后细胞碱性磷酸酶染色及Von Kossa染色呈阳性,成脂诱导后细胞油红O染色呈现阳性.结论 应用全骨髓贴壁法可以分离培养出高纯度的大鼠bMSCs,培养的细胞扩增迅速、生物学特性稳定,是一种较为理想的体外分离扩增bMSCs的培养体系.

Objective To establish a method of isolating and cultivating rat bone marrow mesenchymal stem cells（BMSCs） in vitro and explore the biological characteristics. Methods BMSCs were isolated and purified for culture in vitro and serial passage by the adherent culture method. The morphological changes of the bMSC were continuously observed under the inverted microscope. The cell growth curve was measured by MTT method. The third generation of bMSCs were detected with flow cytometry（FCM）, and incubated in osteogenesis and adipogenesis. Osteogenous potential was assessed by alkaline phosphatase staining and Von Kossa staining. Adipogenic potential was evaluated by Oil red O staining. Results The adherent cells showe spindle shape, polygonal shape and fibroblastcell-like shape and the size of bMSCs were homogeneous. CD29, CD44, CD105 and CD166 memberane antigens could be detected in 97% of the third generation of bMSCs, but CD34, CD80 and CD86 membrane antigens were not detected. Following induction, alkaline phosphatase staining and Oil red O staining produced a strong reaction in cells.Conclusion The whole bone marrow adherent culture is a convenient and efective method to obtain purified bMSCs with strong proliferative ability and capability of multidirectional differentiation.