大鼠骨髓间充质干细胞的分离培养纯化及生物学特性的鉴定

Isolation, Culture and Purification of Bone Marrow-derived Mesenchymal Stem Cells in SD rats and Identification of Its Biological Characteristics

【目的】探讨大鼠骨髓间充质干细胞（rat bonemarrow—derived mesenchymal stem cells，rBMSCs）体外分离、培养和纯化方法，鉴定其生物学特性，为BMSCs应用奠定技术基础。【方法】采用密度梯度离心法结合贴壁分离法分离培养纯化SD大鼠BMSCs，观察其形态，测定细胞生长曲线，流式细胞仪检测细胞表面标记，成骨诱导试剂盒检测rBMSCs向成骨细胞分化的潜能。【结果】显微镜下rBMSCs呈梭形、多角形成纤维细胞样形态，大小均一，漩涡状生长。生长曲线分析rBMSCs贴壁2d基本无增殖，处于潜伏期，对数增殖期为3～7d，d8后进入平台期，细胞出现接触抑制现象。rBMSCs表面标志显示第3代rBMSCs纯度可达97％以上，CD44、CD90呈阳性表达，CD34呈阴性表达；向成骨诱导rBMSCs10d后细胞表现成骨细胞特性，碱性磷酸酶活性明显升高。【结论】本实验建立了一种理想的体外分离扩增纯化rBMSCs的培齐体系。实验结果表明所分离培养的rBMSCs纯度高、生物学特征稳定，适用于对BMSCs进一步的应用研究。

[Objective]To explore the appropriate method of isolation, culture and purification of rat bone marrow-derived mesenchymal stem cells（rBMSCs） in vitro, and to identify its biological properties for further study and clinic application. [Methods]Rat BMSCs were isolated with density gradient centrifugation combined with attachment culture method from bone marrow of the young SD rats. The shape was observed and the growth curve was detected. Flow cytometry was applied to identify the cell membrane antigens. The potential in osteogenous differentiation of rBMSCs was evaluated by the osteogeneic inducer kit. [Results]It was found that rBMSCs were spindle shape, polygonal shape and fibroblast-cell-like shape and the size of rBMSCs was homogeneous under invert-microscope. The morphology of subculture was more homogeneous. The rBMSCs were still in latent phase after being adherent to the bottom for 2 days. And 3-7 days later, cells were in log phase. Eight days later, cells came into platform phase and displayed contact inhibition. CD44 and CD90 membrane antigen could be detected in 97 % of the third generation of rBMSCs, but not CD34 membrane antigen. After osteogeneic inducer was added in vitro for 10 days, the alkaline phosphatase activities were increased obviously. [Conclusion]An effective and appropriate method of the isolation, culture and purification of rat rBMSCs in vitro has been established, rBMSCs isolated and purified by density gradient centrifugation combined with attachment culture method have high purity and proliferation activity. The biological properties are stable.