目的体外分离培养人骨髓间充质干细胞(MSCs)并研究其生物学特性,为进一步实验研究打下基础。方法通过密度梯度离心法及贴壁培养分离纯化人骨髓中的单个核细胞、体外培养传代、倒置相差显微镜观察细胞生长情况、瑞氏染色及透射电镜观察细胞形态及结构、绘制生长曲线、流式细胞仪检测细胞表面标记及增殖周期、免疫细胞化学及细胞化学染色对分离培养细胞进行鉴定。结果原代和传代培养的细胞以成纤维样为主,有较强的生长增殖能力;流式细胞仪检测细胞表面标记:CD29、CD105阳性,CD34阴性;细胞周期分析:90%以上的细胞处于G0/G1期;细胞CD106、纤维连接蛋白表达阳性,CD45、CD14及层连蛋白、胶原蛋白Ⅱ表达阴性,细胞糖原(PAS)染色强阳性,细胞碱性磷酸酶(ALP)染色阴性。表明细胞不是造血细胞且未向成纤维细胞、成骨细胞、软骨细胞分化,而是处于未分化阶段的间充质干细胞。结论通过密度梯度离心法及贴壁传代培养可以分离纯化MSCs以满足下一步实验研究,该细胞具有特殊的生物学特性。 Objective To isolate and culture human bone marrow mesenchymal stem cells (MSCs) in vitro and study their biological characteristics, which lay the foundation for further experimental research. Methods The mononuclear cells of human bone marrow were isolated and purified by density gradient centrifugation and adherent culture. The cells were passaged in vitro and the growth of cells were observed by inverted phase contrast microscope. The morphology and structure of cells were observed by Wright staining and transmission electron microscopy. Cell surface markers and proliferation cycles were detected by cytometry. Immunocytochemistry and cytochemical staining were used to identify the cultured cells. Results Primary and subcultured cells were mainly fibroblast-like, with strong ability of growth and proliferation. Cell surface markers were detected by flow cytometry: CD29, CD105 positive and CD34 negative; Cell cycle analysis: more than 90% G0 / G1 phase. The expression of CD106 and fibronectin was positive. The expressions of CD45, CD14, laminin and collagen Ⅱ were negative. The staining of cell glycogen (PAS) was strongly positive and the staining of cell alkaline phosphatase (ALP) was negative. Indicating that the cells are not hematopoietic cells and did not differentiate into fibroblasts, osteoblasts and chondrocytes, but rather mesenchymal stem cells in an undifferentiated stage. Conclusion MSCs can be isolated and purified by density gradient centrifugation and adherent subculture in order to meet the next experimental study, the cells have special biological characteristics.