一种从骨髓中分离胚胎样干细胞的技术方法  

An isolation method of embryonic-like stem cells derived from adult bone marrow

作　　者：张永云[1] 庞荣清[2] 何洁[2] 赵晶[2] 瞿良[2] 王桂华[2] 潘兴华[2] 张成[3] 

机构地区：[1]云南农业大学农科专业基础实验学示范中心,云南昆明650201 [2]成都军区昆明总医院干细胞与组织器官工程研究中心,云南昆明650032 [3]中山大学附属第一医院神经内科,广东广州510080

出　　处：《中国现代医学杂志》2012年第9期17-21,共5页China Journal of Modern Medicine

基　　金：云南省自然科学基金面上项目(NO.2009ZC151M)

摘　　要：目的建立从成体骨髓中分离胚胎样干细胞的技术方法。方法采用密度梯度离心法从成体骨髓中分离单个核细胞,运用胚胎干细胞扩增用的无血清knockout-DMEM培养基在明胶包被过的培养瓶中扩增培养,光学显微镜下观察细胞形态特征,分别采用免疫荧光染色和RT-PCR方法鉴定多潜能干细胞标志的表达。结果采用无血清培养基在明胶包被过的培养瓶中培养的方法,可从成体骨髓中分离到贴壁生长的细胞,这些细胞与传统方法分离到的间充质干细胞相比体积更小、形态纤细均一,传代后均匀分布生长,不易老化,而且这些细胞弱表达多潜能抗原标志Oct-4、Nanog-3和Sox-2。结论采用扩增胚胎干细胞的技术方法可以从成体骨髓中分离到表达部分多潜能干细胞标志的胚胎样干细胞。【Objective】 To establish a isolation method of embryonic-like stem cells from adult bone marrow.【Methods】 The mononuclear cells were isolated from adult bone marrow by density gradient centrifugation,and were expanded in flask coated-gelatin by serum-free knockout-DMEM medium originally designed for the expansion of human embryonic stem cells.Morphological character of the resulting cells was observed and expression of their pluripotent markers was identified by immunofluorescent staining and RT-PCR analysis respectively.【Results】 The adherent growing cells,weakly expressing Oct-4,Nanog-3 and Sox-2,could be isolated from adult bone marrow.Compared to mesenchymal stem cells isolated by the traditional method,these cells appeared as smaller,morphologically slenderer and homogeneous,and after passage,they grew well-distributed in flask and were not more readily ageing.【Conclusion】 Embryonic-like stem cells expressing a part of pluripotent markers could be isolated from adult bone marrow by the method for the expansion of human embryonic stem cells.

关 键 词：多潜能干细胞 骨髓 分离培养 

分 类 号：R-33[医药卫生]