人骨髓间充质干细胞的分离培养及表型分析  

Culturing in vitro and phenotype of human mesenchymal stem cells

作　　者：梁雪[1] 粟永萍[2] 孔佩艳[1] 陈幸华[1] 彭贤贵[1] 徐辉[2] 常城[1] 艾国平[2] 

机构地区：[1]第三军医大学新桥医院血液科,重庆400037 [2]第三军医大学预防医学院全军复合伤研究所,重庆400038

出　　处：《重庆医学》2005年第9期1328-1330,共3页Chongqing medicine

基　　金：创伤;烧伤与复合伤国家重点实验室开放基金课题资助(2003)

摘　　要：目的体外分离培养人骨髓间充质干细胞(MSCs)并研究其生物学特性,为进一步实验研究打下基础。方法通过密度梯度离心法及贴壁培养分离纯化人骨髓中的单个核细胞、体外培养传代、倒置相差显微镜观察细胞生长情况、瑞氏染色及透射电镜观察细胞形态及结构、绘制生长曲线、流式细胞仪检测细胞表面标记及增殖周期、免疫细胞化学及细胞化学染色对分离培养细胞进行鉴定。结果原代和传代培养的细胞以成纤维样为主,有较强的生长增殖能力;流式细胞仪检测细胞表面标记:CD29、CD105阳性,CD34阴性;细胞周期分析:90%以上的细胞处于G0/G1期;细胞CD106、纤维连接蛋白表达阳性,CD45、CD14及层连蛋白、胶原蛋白Ⅱ表达阴性,细胞糖原(PAS)染色强阳性,细胞碱性磷酸酶(ALP)染色阴性。表明细胞不是造血细胞且未向成纤维细胞、成骨细胞、软骨细胞分化,而是处于未分化阶段的间充质干细胞。结论通过密度梯度离心法及贴壁传代培养可以分离纯化MSCs以满足下一步实验研究,该细胞具有特殊的生物学特性。Objective To isolate and culture the mesenchymal stem celI（MSCs） of human bone marrow in vitro and then study its biological characteristics, Methods To isolate, purify the MNCs from human bone marrow by density gradient centrifugation and then passage them in vitro by adherent culture; observe the cells＇ gowth by inverted phase contrast microscope; observe the cell＇s morphous and structure by Wright＇s staining and transmission electron microscope; detect the cell membrane＇s mark and reproductive cycle by flow cytometer; identify the cultured cells by immunocytochemistry and cytochemical staining methods. Results The primary cells and cultured passage cells mostly were fibroblast cells which had powerful capacity of growth and reproduction. The membrane mark showed that CD29 and CD105 are positive while the CD34 was negative; the cell cycle analysis showed that more than 90 % cells were in the G0/G1 phase; the expression of CD106, fibronection were positive and the expression of PAS was strong positive, the expression of CD45,CD14, laminin,collagen protein Ⅱ and ALP were negative. All the above results shows that the cells are mesenchymal stem cells instead of hematopoietic cell. Conclusion Mesenchymal stem cells can be isolated and cultured by means of density gradient centrifugation and adherent culture, and the mesenchymal stem cells have special biological characteristics.

关 键 词：间充质干细胞 骨髓 细胞培养 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]