大鼠骨髓间充质干细胞的分离培养纯化与鉴定

Isolation,culture,purification and identification of rat bone mesenchymal stem cells

目的:探讨骨髓间充质干细胞(BMSCs)体外获取及培养增殖的方法并鉴定。方法:用密度梯度离心法结合贴壁培养法分离、纯化培养大鼠BMSCs,测定生长曲线,流式细胞仪鉴定,免疫细胞化学检测。结果:密度梯度离心结合贴壁培养法能有效分离纯化大鼠BMSCs,P2、4、6代细胞生长曲线基本一致,增值能力强,呈均一成纤维样细胞,P3代以后细胞均一地表达细胞表面抗原CD44、CD90,不表达CD34,弱表达CD11b/c,细胞表达Connexin43。结论:本实验建立了一种体外稳定培养扩增大鼠BMSCs的方法,培养的细胞成分单一,适用于对BMSCs进一步的应用研究。

Objective： To explore an in vitro practical method of the isolation, culture and purification of bone mesenchymal stem cells （BMSCs） from the bone marrow of the adult rats, and to identify them. Method：BMSCs were isolated with density gradient centrifugation and adherent culture from the bone marrow of the young SD rats. CCK-8 reagent was used to determine the growth curve. Light microscopes were used to study the morpbologic features. Flow cytometry was applied to identify the cells. Immunocytochemistry technique was employed to examine the cell antigens of connexin43. Result： The density gradient centrifugation combined adherent culture can isolate and purify BMSCs eficiently. The similar growth curves of passage2、4 and 6 were exhibiting a large expansive potential and the typical fibroblast-like morphology. The BMSCs are uniformly expressing CD44, C D90, and weakly expressing CD11b/c, and not expressing CD34 after passage 3. Connexin43 are positive in cells. Conclusion： An in vitro method for the isolation and purification of BMSCs from rat bone marrow has been established. The cultured cells were only composed of undifferentiated BMSCs.