人骨髓间充质干细胞体外培养及鉴定

目的：体外分离、培养、鉴定人骨髓间充质干细胞（hBMSC）。方法：利用密度梯度离心法分离出人骨髓单个核细胞（MNC），通过贴壁筛选法建立hBMSC的体外培养体系，并通过一系列检测方法对所培养的细胞进行鉴定。结果：细胞主要呈“成纤维样”，但体积较大、形状不规则、细胞质突起多，细胞核大而疏松，核仁明显。流式细胞仪检测：CD34阳性率为2．09％、CD29阳性率为94．46％；CD54及CD105表达阳性。细胞周期及透射电镜检测提示细胞处于相对原始状态。免疫细胞化学染色显示：CD14、CD45、胶原蛋白Ⅱ呈阴性；CD106、CD166、纤维连接蛋白、波形蛋白及胶原纤维酸性蛋白呈阳性。细胞化学染色：糖原染色呈强阳性；碱性磷酸酶染色呈阴性。通过诱导分化培养可诱导出成骨细胞。结论：成功建立了稳定的hBMSC体外培养体系，按照此培养体系进行培养可获得hBMSC。hBMSC可传代培养，但在培养过程中细胞逐渐出现老化现象，且不能冻存。

Culture and identification of the human bone marrow mesenchymal stem cells in vitro

Objective:To isolate,culture and identify the human bone marrow mesenchymal stem cells(hBMSC). Methods:Human bone-marrow mononuclear cells(MNC) were isolated by centrifugation over Percoll(1.073 kg/L)and cultured in a BMSC culture system in vitro using their adherent characteristics.Many kinds of cellular molecules were measured to identify the cultured cells.Results:The cells show fibroblast-like irregular appearance,many cytoplasm ecptoma,puff and large nucleus with obvious nucleoli.The flow cytometry shows that 2.09% of the cells are CD34 positive,94.46% of the CD34 negative cells are CD29 positive,and CD54 and CD105 are possibly positive.The cell cycle observation and transmission electron microscopy show that the cells are in the primitive status.The immunocytochemistry detection shows that CD14,CD45 and collagen protein II are negative;CD106,CD166,fibronectin,vimentin and collagen fibers acidic protein(GFAP) are positive;staining for glycogen is strongly positive;staining for alkaline phosphatase is negative.The cells can be induced into osteoblast in proper inductive and differentiating culture circumstances.All the above results indicate that the isolation and culture of hBMSC are successful.Although the hBMSC can be serially subcultivated,the cells appear to be aging and they can not be kept by frozen. Conclusion:The steady ex vivo expansion system in which hBMSC can be obtained has been established.