昆明种小鼠胚胎干细胞的分离、培养与鉴定

Isolation,cultivation and identification of embryonic stem cells in Kunming mouse

目的探讨昆明种小鼠胚胎干细胞的分离、培养和鉴定。方法收集小鼠14～16d胚龄的囊胚,以小鼠胚胎成纤维细胞作为饲养层,形成的胚胎干细胞(ES)细胞样集落,分离隆起生长的胚胎内细胞团并继续培养,观察集落的生长状态,通过碱性磷酸酶染色进行的分析鉴定,并对其体外分化能力进行检测。结果共培养收集74个昆明小鼠的囊胚,其中有53个囊胚的内细胞团(ICM)离散程度较强;ES细胞集落的特征呈鸟巢状,其细胞圆小,细胞核大,排列紧密,边缘清晰,细胞之间的界限不清楚;碱性磷酸酶(AKP)染色呈阳性,集落呈红棕色或蓝色,成纤维细胞或分化细胞呈淡黄色或无色;体外悬浮培养时,ES细胞集落能够形成简单拟胚体,且EBs外向性生长,周围分化的细胞形态成多样性。结论昆明小鼠是我国应用最多的实验小鼠,建立昆明小鼠的ES细胞系有利于其转基因动物的获得,能够为我国的科研工作更好地服务。

Objective To probe into the isolation, cuhivation and identification of embryonic stem cells in Kunming mouse. Methods 14 -16 d mouse embryonic blastocysts were collected. Mouse embryonic fibroblast cells were added to the culture as feeder layer. After formation of embryonic stem cell - like colonies, the inner embryonic cell mass was separated and further cuhured. Colony growth was observed and the cell in vitro differentiation was analyzed through alkaline phosphatase staining. Results Blastocysts were collected from 74 Kunming mice, of which 53 blastocysts ICM showed strong degree of dispersion ; embryonic stem cells （ES） colony showed nest - like characteristics. The cells were small round cells, the nucleus were large and tightly packed. AKP positive staining colony was reddish - brown or blue, fibroblast cells or differentiated ceils were light yellow or colorless o In in vitro suspension cuhure, ES colonies can form simple embryoid bodies （EBs）, and EBs showed outward growth pattern and surrounded by differentiated cells with morphological diversity. Conclusion Kunming mice is widely used experimental mice, the establishment of Kunming mouse ES cell lines is beneficial for the acquisition of transgenic animals.