小鼠胚胎干细胞建系及GFP标记

目的：为组织工程提供新型种子细胞,建立胚胎干（ES）细胞系。方法：取129／svj白色小鼠4.5d的囊胚,分离囊胚的内细胞团（ICM）细胞;在条件培养液及小鼠胚胎成纤维细胞（MEF）作铺层下,细胞扩增传代,碱性磷酸酶（AKP）染色及oct4、ssea-1等特异性抗体鉴定细胞,并将细胞注射至裸鼠皮下观察细胞在动物体内的生长变化。结果：细胞体外培养呈“巢状”生长,可以持续传代;AKP染色及oct4、ssea-1等特异性抗体免疫反应均阳性;裸鼠皮下注射的细胞发育生长为包含各胚层细胞成分的畸胎瘤。在此基础上以基因转染方法将细胞标记绿色荧光蛋白（GFP）。结论：从小鼠ICM所分离的细胞为小鼠胚胎干细胞,且标记GFP的ES细胞在传代及形成胚体的过程中均能显示GFP,对将来组织构建的种子细胞起到示踪作用。

Establishment of cell line of mouse embryonic stem cells and its label with GFP

Objective The aging of seed cells is a difficult problem for tissue engineering.In order to supply new types of seed cells for Tissue Engineering,the cell line of Embryonic stem cells (ES) was established.Methods The inner cell mass (ICM) was separated from 129/svj mouse blastocyst of 4.5 days,from which cells were then proliferated and passaged in conditioned medium and on feeder cells(MEF).Special antigen oct4?ssea?1 were used to test these cells,and the latter stained by AKP.These cells were also injected into subcutaneous of nude mice while the in vitro growth can be observed.Results These cells grew as "nests",and proliferated indefinitely in vitro.AKP staining and immunocytochemistry of oct4?ssea?1 were positive.And cells injected into subcutaneous of nude mice grew into teratoma which contains three embryonic layers.After the cDNA of green fluorescence protein(GFP) was transfected into the cells by lipofectine,they showed GFP during passage and embryo formation.Conclusion All these results show the cells we get from mouse blastocyst are mouse embryonic stem cells,and these cells can express GFP after they are transferred with GFP cDNA.