人骨髓间充质干细胞的分离培养及部分生物学特性的实验研究

Experimental study of the culture, isolation and biological properties of human bone marrow mesenchymal stem cells in vitro

目的 建立一种体外分离和培养人骨髓间充质干细胞 (MSCs)的方法 ,并探讨其部分生物学特性。方法 无菌条件下抽取正常健康人骨髓 3～ 5ml,经密度梯度离心得到单个核细胞 ,接种入含 10 %胎牛血清的IMDM培养液 ,测定其生长曲线 ,并观察其贴壁率、细胞周期和超微结构变化。结果 培养的MSCs 3～ 4h开始贴壁 ,贴壁率为 60 %～ 80 % ,2～ 3d即可见集落形成 ,14d左右融合 90 %以上 ,基本上为梭形成纤维细胞状形态 ,流式细胞检测显示有 70 .0 3 %的细胞处于G0 G1 期 ,电镜观察表现出早期细胞的特点。结论 成功地建立了一种分离培养人骨髓MSCs的方法 ,获得的细胞形态单一、生长稳定、增殖较快 ,为进一步应用MSCs促进创伤修复提供了实验基础。

Objective To establish a method for the isolation and culture of human bone marrow mesenchymal stem cells (MSCs) in vitro and explore their biological properties. Methods Mononuclear cells acquired after density gradient centrifugation of the collected bone marrow of 3～5 ml from normal healthy people under aseptic condition were inoculated into IMDM medium containing 10% fetal bovine serum. Growth curve was detected and adhesiveness rate, cell cycle and ultrastructure observed. Results MSCs began to be adhesive after culture for 3～4 h with the rate of 60%～80%. Formation of colony could be observed on the second to third day. More than 90% MSCs, fused at 14th day, were basically fibroblast shaped. About 70.03% of MSCs at G 0/G 1 phase were found by flow cytometry. The ultrastructures of MSCs demonstrated the features of infantile cells. Conclusion The isolated and cultured MSCs by the method successfully established in this study are of singular configuration, stable growth and rapid proliferation. This might provide an experimental foundation for trauma repair.