全骨髓法体外培养分离大鼠骨髓间充质干细胞及PKH26标记

背景：要获得动物实验需要的标记大鼠骨髓间充质干细胞,体外培养、扩增和示踪已成为实验的关键环节。目的：采用全骨髓培养分离大鼠骨髓间充质干细胞,以及PKH26对其体外标记,建立一种方便、实用的分离培养并示踪骨髓间充质干细胞的方法。方法：通过全骨髓培养分离法纯化大鼠骨髓间充质干细胞。经传代扩增,细胞进一步纯化。取第3代大鼠骨髓间充质干细胞按PKH26标记程序进行标记后培养,荧光显微镜下观察标记后细胞生长状态、萤光强度变化和传代培养效果。利用四唑盐比色法测定标记后骨髓间充质干细胞的生长曲线。结果与结论：全骨髓培养分离法能成功获得纯度高的骨髓间充质干细胞,用PKH26标记后的骨髓间充质干细胞呈红色荧光,体外连续传代培养3代后,细胞荧光强度逐渐减弱。PKH26标记骨髓间充质干细胞的生长形态、生长活力不发生改变。结果证实全骨髓培养分离法简便易行,能获取较高纯度的生长增殖状态良好的骨髓间充质干细胞,PKH26荧光标记大鼠骨髓间充质干细胞是一种有效、实用的方法。

Bone marrow mesenchymal stem cells isolated from rats by whole bone marrow adherent culture in vitro and PKH26 labeling

BACKGROUND：In vitro culturing,amplification and labeling are important links to harvest labeled rat bone marrow mesenchymal stem cells（BMSCs） that are satisfactory to animal experiment.OBJECTIVE：To explore a convenient and practical method for separating and labeling BMSCs using whole bone marrow adherent culture,and labeling with PKH26 in vitro.METHODS：BMSCs were isolated and cultivated from the bone marrow of rats by whole bone marrow adherent culture.Further purification was achieved by expansion at serial passages.The passage 3 BMSCs were cultured and labeled with PKH26.The growth,fluorescence intensity and serial subcuhivation of labeled BMSCs were analyzed with fluorescence microscope.The proliferation ability of these labeled cells was tested by MTT.RESULTS AND CONCLUSION：BMSCs were isolated and purified successfully and effectively by the method of whole bone marrow adherent culture.The labeled BMSCs appeared red fluorescence.After 3 passages of serial subcultivation,the fluorescence intensity and the labeling rate of BMSCs were gradually decreased.The biological features such as morphology,growth in vitro were not affected by labeling.BMSCs can be successfully cultivated by whole bone marrow adherence method conveniently.Labeling the BMSCs with PKH26 is an effective and practical method.