昆明种系小鼠胚胎干细胞的分离培养及特性鉴定

目的 提高昆明小鼠胚胎干细胞（ES细胞）建株的成功率。方法 收集小鼠3．5d的囊胚培养，用小鼠胚胎成纤维细胞作为饲养层，形成的ES细胞样集落，经两次亚克隆分离培养ES细胞；进行相差显微镜观察、碱性磷酸酶染色和体内外分化能力鉴定。结果 获得两个稳定ES细胞集落，传至第11代。ES细胞呈集落样生长，碱性磷酸酶染色强阳性，体外分化的细胞沿拟胚体外向性生长，形态多样，在体分化可形成来源于3个胚层的组织。结论 两次亚克地获得的细胞具有ES细胞主要的生物学性状，有助于提高昆明小鼠ES细胞建株的能力。

Isolation,culture and identification of murine embryonic stem cells of Kunming species

Objective To increase the efficiency of establishment of embryonic stem (ES) cell lines from Kunming species mouse. Methods The blastocysts of 3.5 d from Kunming species mouse were collected, cultured on the fibroblast cell feeder layers. The ES cell like colonies were sub clonal cultured twice and cells were observed by phase contrast microscope and identified by AKP staining. The ability of differentiation were examined both in vivo and in vitro . Results Two stable ESC clones were obtained and were passaged to 11th generation. ES cells were colony like, AKP positive. The differentiation of ES cells are outgrowth from embryonic bodies in vitro , while three embryonic derms were formed from ES cells in vivo . Conclusion The cells obtained through twice sub clonal cultures still maintain the main characteristics of ES cell and are helpful to improve the capacity of establishment of ES cell lines.