人骨髓间充质干细胞的分离培养与鉴定

Isolation, cultivation and biological identification of human bone marrow mesenchymal stem cells

目的建立一种简便可行的人骨髓间充质干细胞(hBMSCs)的体外培养扩增方法,并就hBMSCs的表型、细胞周期、生长曲线、超微结构、核型等方面进行初步鉴定。方法应用密度梯度离心法分离hBMSCs,条件培养基培养。相差显微镜下观察hBMSCs形态学变化;流式细胞仪检测hBMSCs的表面标记以及细胞周期;绘制hBMSCs的生长曲线,计算细胞倍增时间;扫描电镜和透射电镜观察hBMSCs的超微结构;Giemsa染色检测hBMSCs的细胞核型。结果密度梯度离心法能分离出纯度较高的hBMSCs。hBMSCs贴壁生长,以长梭形为主。细胞存活率均大于95%。流式细胞仪检测hBMSCsCD29、CD34、CD44、CD45、CD71、CD105、CD166、HLA-ABC、HLA-DR、UEA-1阳性表达细胞比率分别为95.3%、1.8%、94.7%、0.8%、96.2%、96.6%、92.7%、96.3%、1.1%、98.7%。hBMSCs的生长曲线呈S形,在传代7代之前具有较好的生长特性。超微结构显示hBMSCs表面较多突起,孔隙较多,胞质丰富,粗面内质网发达,囊腔扩张,可见大量蛋白分泌物。核型分析显示第3、6代hBMSCs的染色体数量和形态均未发生变化。结论应用密度梯度离心法可得到纯度较高的hBMSCs,能表达hBMSCs的表型特征。hBMSCs能在体外较长期培养,细胞染色体数目未发生改变。

Objective To establish a simple and feasible method to culture and proliferate human bone marrow mesenchymal stem cells （hBMSCs） in vitro, to analyze their biological characterizations such as cell phenotype, generation cycle, growth curve, ultrastructure and caryotype. Methods hBMSCs were isolated by combining density gradient centrifugation with plastic adherence and cultured in specified culture medium. Morphological observations were performed with phase contrast microscope; growth curves of the cells were drawn with help of MTT assay; cell phenotype and generation cycle were detected by flow cytometry; cell ultrastructures were determined by scanning electron microscope and transmission electron microscope, and cell caryotypes were measured by Giemsa staining. Results Higher purity of hBMSCs could be achieved by density gradient centrifugation. The cells displayed long spindle shaped and adherent to the wall. The survival rate was over 95%. The positive expression rates of cell phenotypes were various as followings respectively： CD29, 95.3%; CD34, 1.8%; CD44, 94.7%; CD45, 0.8%; CD71, 96.2%; CD105, 96.6%; CD166, 92.7%; HLA-ABC, 96.3%; HLA-DR, 1.1%; UEA-1, 98.7%. The growth curve ofhBMSCs was ＂S＂ shaped, and the proliferation ability of hBMSCs was strong before 7th passage. The ratio of cells in S＋G2＋M stage of the 3rd and 6th passages was 17.1% and 22.8% respectively. Many erections and pores were observed on cell surface, while abundant cytoplasm, prosperous rough endoplasmic reticulum and plenty of protein excretion were found inside hBMSCs under electron microscopes. The chromosome number at the 3rd and 6th passages were both 46, same as the normal human being＇s. Conclusion Higher purity of hBMSCs can be isolated and cultured by combining density gradient centrifugation with plastic adherence, and cell phenotypes can be maintained as normal human being＇s. The chromosome number ofhBMSCs cultured in vitro for a long time can be kept normal.