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人骨髓间充质干细胞分离与培养方法的建立

Establishment of a method for isolation and cultivation of mesenchymal stem cells from human bone marrow

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【作者】 刘晓丹郭子宽李秀森张双喜毛宁

【Author】 LIU Xiao dan, GUO Zi kuan, LI Xiu sen, ZHANG Shuang xi, MAO Ning (Institute of Basic Medical Sciences,Academy of Military Medical Sciences,Beijing 100850,China) [

【机构】 军事医学科学院基础医学研究所!北京100850

【摘要】 目的 :建立并优化骨髓间充质干细胞 (mesenchymalstemcells ,MSCs)分离纯化及培养扩增的方法。方法 :利用Percoll( 1.0 73g/ml)及Ficoll Hypaque( 1.0 77g/ml)两种分离介质分离骨髓单个核细胞 ,筛选体外培养MSCs适宜的血清及浓度 ,通过流式细胞术分析鉴定MSCs的纯度。结果 :经Percoll分离 ,应用 10 %筛选出的血清培养与扩增的MSCs ,细胞纯度可达 95%左右 ;相反 ,应用常规Ficoll Hypaque分离骨髓单个核细胞或增加血清浓度 ,MSCs纯度显著下降。结论 :建立了一种稳定而实用的体外分离与培养MSCs的方法。

【Abstract】 Objective: To establish a method for isolation and cultivation of mesenchymal stem cells (MSCs) from human bone marrow. Methods: Human bone marrow mononuclear cells were separated by gradient centrifugation on Percoll (density 1.073 g/ml) or Ficoll Hypaque (1.077 g/ml). The cells were incubated in DMEM (low glucose) with 10% or 20% newborn bovine serum from selected lots. The purity of MSCs was analyzed by flow cytometry. Results: The purity of MSCs collected by Percoll and cultured in medium containing 10% NBS was around 95% as assessed by flow cytometry.The purity of MSCs was much lower, nevertheless, when the cells separated by Ficoll centrifugation and / or cultured in medium with 20% NBS. Conclusions: A stable and practical method was established for separation and subsequent cultivation of human bone marrow mesenchymal stem cells. [

【关键词】 间充质干细胞; 骨髓; 细胞,培养的; 流式细胞学;

【Key words】 mesenchymal stem cells; bone marrow; cells,cultured; flow cytometry;

【分类号】R329.24;R331.22;R284.2;

【基金】 国家“九七三”项目资助!课题 (G19990 5 430 2 )

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