以间充质干细胞为饲养层分离培养昆明小鼠胚胎干细胞

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

目的：探讨以间充质干细胞作为饲养层细胞分离培养昆明小鼠胚胎干细胞（Embryonic stem cell，ES细胞）的方法。方法：以鼠间充质干细胞作为饲养层，收集受孕3．5～4d昆明小鼠的囊胚和桑椹胚进行培养，筛选纯化细胞集落，使其稳定传代后，采用光镜，碱性磷酸酶染色及Oct—d染色对其形态学和生物学性状进行初步鉴定。结果：以间充质干细胞作为饲养层细胞分离培养的昆明小鼠ES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，未分化的ES细胞显微镜下为黄褐色，分化的不着色；Oct—d染色阳性。结论：以间充质干细胞作为饲养层细胞分离培养昆明小鼠胚胎干细胞可行，得到的胚胎干细胞能保持特殊的形态和未分化状态。

Objective： By cocculture with mesenchymal stem cells as feeder cells, we observe whether we can isolate and culture embryonic stem cells（ ES cells ） which maintain undifferentiated and multipotential. Methods ： tissues and cell types were used in basical research to cure disease. The blastocysts of 3.5 d from Kunming species mice were collected and cultured on mesenchymal stem cells layers. The embryonic stem cell colonies were preliminarily identified by light microscope AKP staining and Oct-4 staining in morphology and biological character. Results：The embryonic stem cell colonies by coccuhure with mesenchymal stem cells as feeder ceils were identified. The results showed that： 1. all the ES cells had its typical morphological characteristics such as nestlike colonies, round or elliptic, with clear edge and smooth surface, strong refraction , significant swelling growth, compactness of cell aggragation and indistinction between two edges of neighbour cells within a colony, relatively bigger nuclear compared with little cytoplasm, i.e., high ratio of the nuclear-cytoplasm; 2.after the AKP staining, all of the ES cells showed strong AKP activity as same as the ES cells of F0, and on the contrary, the embryonic fibroblast ceils had no expression of AKP showing the lost of staining;3, after the Oct-4 staining, all of the ES cells showed strong Occt-4 activity. Conclusion：By cocculture with mesenchymal stem cells as cells,we can isolate and culture embryonic stem cells （ES cells） which maintain undifferentiated and muhipotential.