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目的:建立体外分离、培养及纯化大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的方法,并对细胞进行形态学观察、表面标志物鉴定及分化潜能检测,为大鼠MSCs进行细胞移植治疗脑损伤的研究奠定基础。方法:用全骨髓贴壁培养法分离、培养、纯化大鼠MSCs,倒置相差显微镜进行形态学观察,流式细胞仪检测细胞表面标记物,定向诱导向成脂、成骨方向分化。结果:72小时首次全量换液7天后观察可见呈放射状排列的细胞集落,以梭形细胞为主,细胞规则、生长旺盛,可连续传代10代以上;取3代MSCs流式细胞仪检测,CD90呈阳性表达,CD34、CD45均呈阴性表达;细胞传代后加入成脂、成骨诱导剂定向诱导分化,油红O染色及茜素红染色均呈阳性。结论:全骨髓贴壁筛选培养法可大量分离、纯化、扩增大鼠MSCs。经诱导鉴定MSCs具有多项分化潜能。
OBJECTIVE: To establish a method for the isolation, culture and purification of rat mesenchymal stem cells (MSCs) in vitro. Morphological observation, surface marker identification and differentiation potential assay were performed on the cells for rat MSCs transplantation The treatment of brain injury lay the foundation for the study. Methods: MSCs were isolated, cultured and purified by whole bone marrow adherent culture. Morphological observation was performed by inverted phase contrast microscope. Cell surface markers were detected by flow cytometry and differentiated into adipogenic and osteogenic directions. Results: After 7 days of the first full volume change of liquid for 72 hours, we can see that the cells arranged in a radial arrangement were dominated by spindle cells with regular growth and vigorous growth, which could be passaged for more than 10 generations continuously. After passage 3 MSCs were detected by flow cytometry, CD90 The positive expression of CD34 and CD45 were negative. The cells were passaged into adipocytes and induced by osteogenic inducer. Oil red O staining and alizarin red staining were all positive. Conclusion: Whole bone marrow adherent culture can be used to isolate, purify and amplify rat MSCs. Induction MSCs identified with a number of differentiation potential.