目的 建立猪骨髓间质干细胞(bone marrow mesenchymal stem cells,BMMSC)体外分离培养、纯化的方法,观察其生物学特性,为其作为组织工程学种子细胞提供实验基础.方法 取猪的胸骨骨髓6～8ml,用Ficoll淋巴细胞分离液提取单核细胞,采用贴壁法培养.接种后2h利用倒置显微镜观察细胞的大体形态,并利用流式细胞仪检测细胞抗原表达.结果 原代培养接种3～4h后细胞开始贴壁,24h后贴壁细胞数量明显增多,3～4d后细胞呈纺锤形且形成为单个或数个细胞克隆,7～9d后集落迅速增多,细胞融合,在10～12d细胞铺满全层.流式细胞仪监测到BMMSC表达CD29、CD44、CD105、KDR,不表达HLA-DR、CD11a、CD14、CD31、CD34、CD45.结论 采用密度梯度离心法培养猪的BMMSC生长稳定,增殖能力强,具有间质干细胞的表面抗原特征,该方法可作为培养猪BMMSC的常规方法.%Objective To establish the methods of isolation, expansion, and purification of porcine bone marrow mesenchymal stem cells (BMMSC) in vitro, and provide the experimental basis for BMMSC as tissue engineering seed cells by observing its biology characteristic. Method A volume of 6 -8ml porcine bone marrow was aspirated from sternum. The mononuclear cells were harvested using Ficoll lymphocyte separating medium at a density of 1. 077. the BMSC were cultured by the adherence method. Cellular morphology was observed by microscope, and cellular surface antigen expression was examined by the instrument flow cytometry. Result After seeded 3 -4 hours, cells adhered to the bottom. Twenty-four hours later, the number of the adhered cells significantly increased, and 3 -5 days later, the primary cultured cells were in uniformly long spindle-shaped form and formed cell-colony. After seeded 7 -9 days, the cell-colonies rapidly increased,and 10- 12 days later, cells spread to the bottom. The test of Flow cytometry showed that the BMMSC were positive for CD29,CD44,CD105 and KDR, while negative for HLA- DR, CD11a, CD14, CD31, CD34 or CD45. Conclusion By density gradient centrifugation and adherence method, BMMSC of porcine were obtained easily and proliferate rapidly, which showed the antigen characteristics of mesenchymal stem cell. The proposal method could be used as a routine method for B MMSC preparation.
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