成人骨髓间充质干细胞的体外培养和表型鉴定  

Isolation in vitro cultivation and phenotype identification of human bone marrow-derived mesenchymal stem cells

作　　者：柳向东[1] 柴冈[1] 李东[1] 崔磊[1] 刘伟[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学第九人民医院整复外科上海市组织工程研究与开发中心,上海200235

出　　处：《上海第二医科大学学报》2004年第4期302-306,共5页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：国家重点基础研究发展规划资助项目("973"项目)(1999054300);上海第二医科大学博士点建设基金资助项目(BXJ0205).

摘　　要：目的 建立成人骨髓间充质干细胞(hMSCs)的分离培养方法,并对其表面标志进行检测,研究骨髓间质干细胞(MSC)基本生物学特性。方法 采用Percoll(1.073 g/mL)梯度离心分离hMSCs,体外扩增,流式细胞仪检测hMSCs表面抗原表达。结果成人骨髓间质干细胞在体外扩增原代及传代培养显示,hMSCs具有活跃增殖的能力,每10 mL骨髓可获得(1.25±0.56)×106个原代细胞,7代可获得(5.32±1.15)×1010个细胞(n=5),随着传代次数的增加,细胞的增殖能力下降。流式细胞仪检测生长良好的第3代hMSC显示CD29、CD44、CD106、CD105、CD166、HLA-A/B/C表达阳性,CD34、CD45、CD14、CD31、CD49d、CD54、HLA-DR/DP/DQ表达为阴性。细胞周期分析显示,hMSCs中,S+G2+M期的细胞约占(16.25±3.18)％,其中s期为(4.12±2.35)％;而处于G0+G1期的细胞总数占(76.35±5.28)％,说明大部分细胞仍然处于静止期。流式细胞仪检测表明分离的hMSCs具有间充质干细胞的特征。结论 hMSCs有很强的自我更新能力,在体外传代培养可维持良好的干细胞生物学特性,为进一步深入研究hMSCs作为组织工程种子细胞的应用提供参数。Objective To establish a method for isolation and cultivation of human bone marrow-derived mesen-chymal stem cells (hMSCs) and to identify their surface makers. Methods hMSCs were isolated from bone marrow and purified via Percoll (1.073 g/mL) gradient centrifugation and culture in vitro, the proliferation and growth characteristics of hMSCs were observed during primary and passage cultures. hMSCs were analyzed by the flow cytom-etry to detect the surface antigens. Results hMSCs had an active proliferative ability in primary and passage cultures. In primary culture approximately(1. 25±0. 56)×106 cells per 10 mL were obtained. At passage7, isolated cells could be expanded to (5.32±1.15)×1010 cells (n =5) . Flow cytometry analysis showed that about 80 % of hMSCs was in the resting phase (G0 + G1) of cell cycle. The analysis also demonstrated that in vitro expanded hMSCs expressed mesenchymal cell marker, including CD29、CD44、CD106 、 CD105、CD166 、HLA-A/B/C. However they didn't express CD34、CD45、CD14、CD31、CD49d、CD54、HLA-DR/DP/DQ. Conclusion hMSCs have the self - renewal capacity and can maintain the phenotypes of mesnchymal stem cell during in vitro culture. Thus hMSCs have a great potential as the seed cells for tissue engineering.

关 键 词：骨髓间充质干细胞 体外培养 细胞表型 鉴定 组织工程 流式细胞仪 生物学特性 

分 类 号：Q813.12[生物学—生物工程]