人骨髓间充质干细胞分离培养及向软骨细胞定向分化的实验研究

目的：探讨人骨髓间充质干细胞体外分离培养和向软骨细胞定向分化的条件，为髁突软骨组织工程提供种子细胞。方法：采用Percoll分离液，根据细胞密度梯度原理，从健康人骨髓组织中分离骨髓间充质干细胞，对所获得的细胞进行体外培养和表面标志的流式细胞仪分析。对经过鉴定证实的骨髓间充质干细胞用含胰岛素、转铁蛋白、丙酮酸、地塞米松和TGF－β的诱导培养基处理7－14d。对诱导细胞进行蕃红花O－亮绿染色和Ⅱ型胶原染色，鉴定诱导后细胞的软骨细胞表型，结果：培养的人骨髓间充质干细胞均一表达CD29、CD44，而CD34、CD45和HLA－DR呈阴性。细胞经过诱导液作用14d后，蕃红花O－亮绿染色和Ⅱ型胶原染色阳性。结论：采用比重为1073g/L的Percoll能分离获得高纯度的人骨髓间充质干细胞（纯度大于95％）。该细胞经诱导液作用，可定向分化为软骨细胞。

Experimental study of the isolation,culture and in chondrogenic differentiation of human bone mesenchymal stem cell

OBJECTIVE: To study the isolation of human bone marrow mesenchymal stem cells (MSCs) and in vitro differentiation into chondrocytes as potential seed cell for condyle cartilage tissue engineering. METHODS: Human MSCs were isolated by percoll solution from normal human bone marrow sample and cultured in flasks. Specific cell surface markers were identified by flow-cytometry. After the cells were treated with inductive medium containing insulin, transferrin, pyruvate, dexathemesone and TGF-beta for 7 - 14 days, microscopic, histological and immuno-histo-chemical studies were performed for chondrogenic phenotype identification. RESULTS: Primary cultures of human MSCs express CD29 and CD44 positively and meanly, but CD34, CD45 and HLA-DR negatively. After 14 days of induction, the cells were positively stained by safranin O. Immunohistochemical analysis proved strong type II collagen expression. CONCLUSIONS: Percoll helps to generate a better isolation of MSCs from human bone marrow aspirates with a purity more above 95%. The isolated MSCs can be expanded and induced in vitro to differentiate into chondrocytes by inductive medium.