人骨髓间充质干细胞的分离、培养与鉴定

Isolation, culture and identification of human mesenchymal stem cell

目的：探讨密度梯度离心结合贴壁筛选法分离培养人骨髓间充质干细胞（MSCs）的条件，为组织工程选择合适的种子细胞奠定基础。方法：采用密度梯度离心法将人MSCs自少量骨髓中分离并培养，观察其增殖和生长特性，绘制生长曲线，测定贴壁率，免疫组化与流式细胞仪细胞表型鉴定。结果：人MSCs生物性状稳定，不同代次生长曲线相似，第5～6d细胞数目最多；传代后10h贴壁率高达90％。MSCs表达CD44、CD90，但不表达CD34、CD45。结论：密度梯度离心法是分离人MSCs的理想方法，MSCs能为组织工程提供足量种子细胞。

Objective： To investigate isolating and culturing conditions of human mesenchymal stem cells （hMSCs） in vitro by density gradient separation method for selecting suitable bone tissue engineering seed cells. Mothoda： hMSCs were isolated by density gradient separation method and cultured. The proliferation and growth characteristics of primary and passage cells were studied by the detection of growth curve and adhesion rare, while flow cytometry and immunohistology methods were used to identify the phenotypes of cultured cells. Rosults： hMSCs grew well in vitro, with the similar growth curve among different passage cells. The number of cells reached the peak at the 5 and 6 days. Most of cells adhered to the wall within 10 hours culturing, hMSCs were positive for CD44 and CD90 ,however, negative for CD34 and CD45. Conelusions： The density gradient separation method is an ideal way for isolating of hMSCs, which is reliable to obtain enough seed cells for bone tissue engineering research.