昆明种小鼠胚胎干细胞的培养及鉴定

Isolation,Culture and Identification of Murine Embryonic Stem Cells of Kunming Species

目的从昆明种小鼠的早期胚胎中获取并培养胚胎干细胞。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,72h后分离隆起生长的内细胞团并继续培养,观察集落的生长状态,并通过碱性磷酸酶染色进行鉴定。结果ES细胞有其典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大;对ES细胞碱性磷酸酶进行检测,在AKP底物作用下,未分化的ES细胞显微镜下为棕褐色,分化的不着色。结论昆明种小鼠囊胚在小鼠胚胎饲养层细胞上可以发育为胚胎干细胞。

Objective To isolate and culture embryonic stem （ES） cells from Kunming species mouse. Methods The blastocysts of 3.5 d from Kunming species mouse were collected, cultured on the fibroblast cell feeder layers for 72 h. The cells from inner cell mass （ICM） were isolated and subsequently cultured in vitro. Then the stem cells were identified by AKP stainin Results The ES cells had its typical morphological characteristics such as nest - like colonies, round or elliptic, with clear edge and smooth surface, strong refraction, significant swelling growth, compactness of cell aggregation, and obscure distinction between two edges of neighbour cells within a colony, relatively bigger nucleus compared with little cytoplasm. After the AKP staining, all of the ES cells showed strong AKP activity, and on the contrary, the embryonic fibroblast cells had no expression of AKP showing. Conclusions The embryonic stem cells can be isolated and cultured from Kunming species mouse.