影响人类胚胎生殖细胞分离克隆的因素  

Influencing Factors on the Efficiency of Isolation and Culture of Human Embryonic Germ(EG) Cells

作　　者：华进联[1] 窦忠英[1] 徐小明[1] 李松[1] 杨玉艾[1] 雷安民[1] 

机构地区：[1]西北农林科技大学陕西省干细胞工程技术研究中心,陕西杨凌712100

出　　处：《农业生物技术学报》2004年第3期294-299,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30200137;39570363);国家重点基础研究发展规划(973)(No.G199954301);国家高技术研究发展计划(863)(No.2002AA216161)基金资助

摘　　要：探讨胚龄培养液、细胞因子、饲养层细胞等因素对人类胚胎生殖细胞分离克隆的影响。以4～13周龄流产胎儿生殖嵴或性腺为材料，小鼠胎儿成纤维细胞(MEF)、人胎儿成纤维细胞(hEF)、STO细胞和牛胎儿成纤维细胞(bEF)作饲养层，高糖DMEM(Dulbecco's minimum Eagle’s medium)为基础培养液，添加10^-4mol／L 2Me(2-巯基乙醇)、胎牛血清(FBS)或新生牛血清(NBS)、白血病抑制因子(LIF)、碱性成纤维细胞生长因子(bFGF)、干细胞冈子(SCF)等为培养液，探讨影响人类胚胎生殖细胞分离克隆的因素。结果表明7～12周流产儿适于人类胚胎生殖细胞的分离克隆；人类胚胎生殖细胞体外培养以添加15％FBS为宜：添加4ng／mL LIF+4ng／mL bFGF+20ng／mL SCF能明显改善人类胚胎生殖细胞的分离克隆；MEF、STO、bEF和hEF可提高人原始生殖细胞(PGCs)的贴壁率，能促进PGCs源的人类胚胎干细胞(ES)的分离效率；以MEF作为饲养层效果较好。Embryonic germ (EG) cells are pluripotent cells derived from primordial germ cells(PGCs) of gonads, gonadal ridges and mesenteries, analogies of fetuses,with the ability to undergo both highly self-renewal and multiple differentiation. These cells can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cells of the body. The paper investigated some influencing factors on the efficiencies of isolation and culture of human EG cells,such as the ages of fetuses, serum, cytokines and feeder cells. The results demonstrated that 7~12 weeks of fetuses were optimal for in vitro culture of human EG cells. The basic medium consisted of DMEM(Dulbecco's minimum Eagle's medium),supplemented 1×10-4 mol/L non-essential amino acid, 2×10-3 mol/L L-glutamine and 1×10-3 mol/L pyruvate. Basic medium supplemented 15%FBS(fetal bovine serum), 4 ng/mL LIF(human recombinant leukemia inhibitory factor), 4 ng/mLbFGF(basic fibroblast growth factor) and 20 ng/mL SCF could clearly improve the efficiencies of isolation and culture human EG cells. Feeder cells with murine embryonic fibroblasts(MEF) were better than that with human embryonic fibroblasts(hEF), or STO or bovine embryonic fibroblasts (bEF).

关 键 词：人类 胚胎生殖细胞 分离 克隆 原始生殖细胞 胚胎干细胞 

分 类 号：Q813[生物学—生物工程] Q78