改良法分离培养大鼠骨髓间充质干细胞的实验研究

IMPROVED ISOLATION AND CULTURE OF RAT BONE MARROW-DERIVED MESENCHYMAL STEM CELLS

目的:改良大鼠骨髓间充质干细胞(MSCs)原代分离方法,探讨大鼠MSCs体外培养的适宜条件。方法:比较改良与传统单纯贴壁培养法、密度梯度离心法结合贴壁分离法分离培养大鼠MSCs,观察不同方法获得原代细胞形态及不同代MSCs的生长曲线,流式细胞仪检测细胞表面标记,同时观察不同浓度的胎牛血清(FBS)对MSCs生长曲线的影响。结果:改良单纯贴壁培养法获得的原代细胞贴壁时间早、生长快,与其他两种分离方法差异明显(P<0.01);原代MSCs用15%的FBS培养,其增殖明显高于5%、10%及20%的FBS培养;第3代以后MSCs用5%的FBS培养有力于细胞纯化。结论:成功地改良了分离培养大鼠MSCs的方法,配合不同血清浓度培养基可使收获MSCs效率增高。

Objective： To improve isolation and culture of rat bone marrow-derived mesencnymai stem cell and explore an appropriate condition for cell culture. Methods： Rat MSCs were isolated by improved and traditional non-adherent methods and Pereoll gradient centrifugation. The shape and growth curve of MSCs were observed. The growth curve of MSCs in different concentration of fetal bovine serum （FBS） was observed at the same time. Result： It was found that the improved method can obtain more adherent cells which grow fast than with the other methods （P〈0.01）. The proliferation of primary MSCs with 15% FBS were increased compared to those with other concentrations of FBS. MSCs were purified with 5% FBS after the 3rd passage. Conclusion： A successful method improves the isolation and subsequent cultivation bone marrow-derived MSCs. Cultivating with the different concentrations of FBS can help to obtain stable and active MSCs.