目的 探讨骨髓间充质干细胞(mesenchymal stem cells，MSCs)理想的分离、培养方法。方法 采用密度梯度离心法和全骨髓贴壁法分离培养人骨髓干细胞，并将其分别置于普通培养瓶、60Co培养瓶中培养，应用倒置显微镜和免疫组化法观察和鉴定骨髓间充质干细胞；流式细胞术检测细胞表面标志。结果 人骨髓干细胞呈均一梭形、成纤维细胞样，形成集落样生长。60Co培养瓶细胞贴壁情况明显好于普通培养瓶，且费用低；免疫组化及流式细胞术显示CD34、CD45表达为阴性， CD29、CD44表达为阳性。60Co照射处理的培养瓶结合采用全骨髓贴壁法分离培养法获得的骨髓干细胞活性高，增殖力强，克隆形成早，传代时间短。结论 全骨髓贴壁法和密度梯度离心法均可获得高纯度贴壁生长的骨髓干细胞,其中60Co照射处理的培养瓶结合采用全骨髓贴壁法分离培养简单易行，骨髓干细胞增殖快，活性好，传代力持久。可以收获大量、高纯度的MSCs；本实验为MSCs进一步的研究与临床应用提供了实验方法及依据。
Abstract
Objective To seek better methods of isolating and culturing bone mesenchymal stem cells(BMSCs)．Methods BMSCs were isolated from human bone marrow by density gradient centrifugation and adherence separation respectively. BMSCs cultured in normal culture flasks and flasks irradiated by 60Co were observed. The characteristics and morphology of BMSCs were observed with an inverted microscope each day. The surface markers of BMSCs were identified by immunocytochemical technique. The expression of surface molecules was examined by flow cytometry. Results BMSCs were in uniformly long spindle shaped and cell colonies were formed. Culture flasks irradiated by 60Co (5 Gy) were better for the adherence and growth of BMSCs. Immunocytochemistry and flow cytometric analysis indicated that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. The primary cells cultured by adherence separation with flasks irradiated by 60Co showed higher cytoactivity, faster proliferation, earlier colony confluence and a shorter time for passage. Conclusions BMSCs are obtained both by density gradient centrifugation and adherence separation. Adherence separation with flasks irradiated by 60Co is an efficient technique for the isolation and purification of BMSCs.
关键词
骨髓间充质干细胞 /
细胞培养
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基金
国家973项目基金资助（2005CB522603）；武警部队资助项目（2006006）
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