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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

【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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