影响脐血多能非造血成体干细胞分离相关因素探究.doc

影响脐血多能非造血成体干细胞分离相关因素探究 　 作者：李云涛，徐燕，孟恒星，葛薇，李桥川，万长春，于珍，李长虹，邱录贵 【摘要】 　　为了研究低血清条件下从脐血中分离多能非造血成体干细胞的相关影响因素，探索优化培养条件，通过不同培养液、接种密度、首次换液时间对脐血多能非造血成体干细胞生长的影响的比较，并以选定的条件对其培养扩增，进行免疫表型及诱导分化能力检测。结果表明：在低血清条件下，DMEM/F12培养液更适合于脐血多能非造血成体干细胞生长； 1×106／cm2是原代培养的适宜接种密度，原代第72小时首次换液为最佳换液时问。利用所选条件培养的细胞可在体外持续扩增，并具有良好的分化潜能。结论：建立了人脐血多能非造血成体干细胞的体外优化培养条件，为其在组织工程方面的应用作出了新的探索。 【关键词】 干细胞； 多能非造血成体干细胞； 脐血； 分离效率 　　Related Factors Affecting the Isolation of Multipotent Non-hematopoietic Adult Stem Cells from Umbilical Cord Blood 　　　Abstract To investigate the related factors affecting the isolation of multipotent non-hematopoietic adult stem cells (MNASCs) from human umbilical cord blood in low serum (2%) condition，the isolation conditions were optimized and the yield of MNASCs was improved. MNASCs from human umbilical cord blood samples were isolated，and the effects of medium component，medium exchange time and initial plating density for isolation of MNASCs were studied. Then，the MNASCs were isolated and cultured in optimal condition，the surface antigen expression and differentiation potential of MNASCs were detected. The result showed that the medium of DMEM/F12 was better than IMDM and DMEM-LG for MNASCs culture in low serum condition. The optimal yield of MNASCs was obtained when mononuclear cells were cultured at a initial plating density of 1×106 cells／cm2 and the medium was exchanged to remove the nonadherent cells after 72 hours of inoculation. MNASCs isolated and cultured under the above-mentioned conditions maintained a homogenous morphology，high potential ability of expansion and differentiation. It is concluded that culture conditions with low serum defined in this study is optimal for the successful isolation and expansion of umbilical cord blood MNASCs with high numbers for subsequent cellular therapeutic approaches. 　　Key words stem cell; multipotent non-hematopoietic adult stem cell; umbilical cord blood; isolation efficiency 近年来一些研究发现，脐血中存在具有多向分化潜能的非造血成体干细胞(multipotent non-hematopoie