大鼠骨髓间充质干细胞体外分离培养的实验研究

Isolation and culture of rat bone marrow-derived mesenchymal stem cells in vitro

目的:探讨大鼠骨髓间充质干细胞(MSCs)体外分离、培养的适宜条件。方法:采用密度梯度离心法结合贴壁分离法分离培养大鼠骨髓MSCs,观察其形态,流式细胞仪检测细胞表面标记,MTT法观察不同浓度的胎牛血清(FBS)对MSCs生长曲线的影响及不同代MSCs的生长曲线和细胞贴壁率。结果:大鼠MSCs呈梭形或纺锤形,CD34、CD45表达阴性,CD44表达71.5%、CD166表达83.2%;MSCs用10%的FBS培养,其增殖明显高于用无血清、5%及20%的FBS培养;第1、3、5、10代MSCs的生长曲线及细胞贴壁率无明显差异(P>0.05)。结论:密度梯度离心法结合贴壁分离法适于大鼠MSCs的分离;10%的FBS适合MSCs的培养;早期传代对MSCs的增殖无影响。

Objective： To explore an appropriate condition of isolation and culture of rat bone marrow-derived mesenchymal stem cells （MSCs） in vitro, Methods： Rat MSCs were isolated with percoll and nonadherent ceils were discarded 48 hours later. The shape was observed and CDa4, CD44, CD45, CD166 were detected with Flow cytometry analysis. The growth curve of MSCs in different concentration of fetal bovine serum （FBS） was observed by MTT colorimetry. The growth curve and attachment efficiency of MSCs of passage 1（P1）, P3, P5 and P10 were also observed. Results： It was found that most of MSCs were shuttle or spindle shape. The expression of CD44 and CD166 were 71.5% and 83.2% and CD34 and CD^45 were negative, The proliferation in the fourth to the sixth day with 10% FBS were increased than those with other concentrations of FBS. The growth curve and attachment efficiency of MSCs of P1, P3, P5 and P10 were found no different （P 〉 0.05）, Conclusion： Pereoll and anchoring methods were appropriate for the isolation of MSCs and 10% FBS was more suitable for the culture of MSCs, Early passaging did not affect the proliferation of MSCs.