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

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.