体外培养获得稳定高效胚胎干细胞滋养层的实验研究  

Experimental Study on Stable and Effective Condition of Feeder Layer for the Culture of Embryonic Stem Cell in vitro

作　　者：杨卫兵[1] 刘尧[2] 李欣[1] 秦茂林[2] 李红丽[2] 

机构地区：[1]解放军181医院皮肤科,广西桂林541002 [2]第三军医大学基础医学部组织学与胚胎学教研室

出　　处：《华南国防医学杂志》2010年第6期439-442,共4页Military Medical Journal of South China

基　　金：广西科学研究与技术开发基金项目（0592007-1G）

摘　　要：目的筛选并优化获得稳定的胚胎干细胞（embryonic stem cells,ESCs）滋养层细胞的来源及其体外培养的最佳条件,为胚胎干细胞体外存活提供必要条件。方法分别取人工流产胎儿皮肤组织、昆明小鼠孕12.5、14.5、18.5d胚胎和新生鼠（P0）组织,分离培养原代胚胎成纤维细胞（embryonic fibroblasts,EFs）并传代制成滋养层。采用活细胞拒染法和免疫细胞化学染色等技术,观察并比较两种来源以及不同时相来源制备的滋养层细胞的功能状态差异。结果细胞活性状态：①人胚来源第1～5代成纤维细胞活性均好;但1～2代时杂细胞较多,此后可见深染色细胞逐渐增多。②E12.5d、E14.5d鼠胚来源的各代细胞均较同代E18.5d和P0来源的活性细胞数目高（P〈0.05）。以E14.5d第3～6代细胞最易培养存活。P0来源第6代与E14.5d来源同代细胞相比,深染变形细胞增多达30%。细胞分泌功能：①人胚来源第3～5代细胞I型胶原蛋白免疫阳性细胞数目少且染色较淡。②鼠胚来源的以E12.5d、E14.5d、E18.5d的第3～6代细胞免疫阳性细胞数目最多,且染色深;P0来源第6代细胞免疫阳性细胞染色深浅不一。结论采用人胚胎来源的成纤维细胞的活性尚可,但分泌功能稳定性欠佳。鼠胚E12.5～18.5d来源的第3～6代成纤维细胞,具有细胞活性强、分泌功能旺盛的特点,是作为胚胎干细胞赖以存活滋养层的最佳选择。Objective To screen the stable and effective condition of feeder layer for the culture of embryonic stem cell in vitro and to supply the necessary condition for in vito survival of embryonic stem cell.Methods Human primary embryonic fibroblasts（EFs） were prepared from skin tissue of abortion fetus.Mouse primary EFs were sterilely prepared from mouse fetus of different embryonic ages（E12.5d,E14.5d,E18.5d） and P〈0 postnatal mouse.Staining of living cells and immunocytochemistry staining were used to observe the differences between these two kinds of cells.Results（1）The cell viability：① Human primary EFs of the first five generations had good cytoactivity.The isolation was not very well in the first two generations.Aging cells were gradually increased into 52.4%,and there were 45.3% dark dying death cells in the sixth generation.② EFs derived from E12.5d and E14.5d mouse had higher number of active cells than those from E18.5d and P〈0 mouse of the same generation（P〈0.05）.The EFs from E14.5d mouse in the third to sixth generations were most easy to be cultured.The dark dying death cells of the sixth generation of EFs from E18.5d and P〈0 mouse were 30% more than those from E14.5d mouse（P〈0.05）.（2）Secretion function of cells：①The numbers of positive cells by I-type collagen immunocytochemistry staining from human EFs of the third to fifth generations were small and the dying was light.②Most positive cells and dark dying were observed in EFs from the third to sixth generation,derived form E12.5d,E14.5d and E18.5d mouse by I-type collagen immunocytochemistry staining.The staining of the sixth generation of EFs derived from P〈0 mouse was inconsistent.Conclusion The viability of human EFs is not bad but their secretion function is not stable.EFs derived from E12.5d-18.5d mouse of the third to sixth generations have good cytoactivity and secretion function which may be the optimal origin of feeder layers for the culture of embryonic stem cell in vitro.

关 键 词：胚胎成纤维细胞 胚胎干细胞 滋养层 

分 类 号：R321-33[医药卫生—人体解剖和组织胚胎学]