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目的探讨体外分离、培养与鉴定大鼠骨髓间充质干细胞(BMSCs)的方法。方法采用贴壁法培养大鼠BMSCs,倒置相差显微镜观察细胞形态,绘制细胞生长曲线;流式细胞仪检测细胞周期、细胞表面抗原标志物;成骨与成脂诱导液诱导BMSCs向成骨细胞与脂肪细胞分化。结果 BMSCs贴壁生长,呈均一长梭形,生长曲线呈“S”型。细胞周期测定大部分处于G1/G0期。P3代细胞高表达抗原CD29、CD44,基本不表达CD45抗原,符合BMSCs表面标志物特征。成脂诱导液诱导BMSCs分化21 d后,油红O染色可见胞内脂滴出现;成骨诱导28 d后,茜素红染色可见连成一片的红色钙结节。结论采用贴壁培养法可纯化、扩增BMSCs;BMSCs细胞表面抗原标志物特性为稳定表达CD44、CD29,不表达CD45;BMSCs具有成骨和成脂分化潜能。
Objective To investigate the method of isolation, culture and identification of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods BMSCs were cultured by adherent method. The morphological changes of cells were observed by inverted phase contrast microscope. The cell growth curve was drawn. Cell cycle and cell surface antigen markers were detected by flow cytometry. Osteoblasts and adipogenic medium induced osteogenic differentiation of BMSCs into osteoblasts Adipocyte differentiation. Results BMSCs adherent growth, showing a uniform long spindle, the growth curve was “S ” type. The majority of cell cycle assays are in the G1 / G0 phase. P3 generation of high expression of antigens CD29, CD44, basically did not express CD45 antigen, in line with the characteristics of BMSCs surface markers. After adipogenic induction induced differentiation of BMSCs for 21 days, intracellular lipid droplets appeared after staining with oil red O. After 28 days of osteogenic induction, alizarin red stained red calcium nodules were observed. Conclusion The adherent culture method can be used to purify and expand BMSCs. The characteristics of BMSCs surface antigen markers are stably expressing CD44, CD29, but not CD45. BMSCs have osteogenic and adipogenic potential.