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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～ 1 4d。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CD2 9、CD44 ,而CD34、CD45和HLA DR呈阴性。细胞经过诱导液作用 1 4d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 1 0 73g/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
Objective To investigate the conditions of human bone marrow mesenchymal stem cells (MSCs) isolated and cultured in vitro and directed to chondrocytes, and to provide seed cells for condylar cartilage tissue engineering. Methods Percoll separation fluid was used to separate bone marrow mesenchymal stem cells from healthy human bone marrow cells according to the principle of cell density gradient. The obtained cells were cultured in vitro and analyzed by flow cytometry. Bone marrow-derived mesenchymal stem cells proved to be authenticated were treated with induction medium containing insulin, transferrin, pyruvate, dexamethasone and TGFβ for 7 to 14 days. The induced cells were bright green light-safflower O staining and type Ⅱ collagen staining to identify the induced chondrocytes phenotype. Results The cultured human bone marrow mesenchymal stem cells expressed CD2 9 and CD44 homogeneously, but negative for CD34, CD45 and HLA DR. After induced by liquid for 14 days, the cells were bright green and type II collagen positive. Conclusion High purity of human bone marrow mesenchymal stem cells (greater than 95% purity) can be isolated using Percoll with a specific gravity of 1073g / L. The cells induced by the role of liquid can be differentiated into chondrocytes