【作者】 王志勇；

【导师】 刘发央； 杜若甫； 徐玖瑾；

【作者基本信息】 甘肃农业大学 ， 草业科学， 2004， 硕士

【摘要】 本实验利用ICR品系小鼠13.5dpc的胚胎制备小鼠胚胎成纤维细胞，并以小鼠胚胎成纤维细胞作为饲养层；收集3.5dICR小鼠的囊胚和桑椹胚进行体外培养，筛选纯化ES细胞集落，使其稳定传代后，对其形态学和生物学性状进行初步鉴定。结果表明：1. 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。2. 对ES细胞碱性磷酸酶进行检测，在AKP底物NBT、BCIP作用下，未分化的ES细胞显微镜下为黄褐色，分化的不着色。3. 通过核型鉴定，ES细胞具有正常的二倍体核型。4. 在有滋养层未加入小鼠白血病抑制因子的条件下，小鼠ES细胞在F2代时将会分化成其它细胞；在有滋养层且加入小鼠白血病抑制因子时，生长至F8代及F11代又会发生分化。更多还原

【Abstract】 The blastocysts of 3.5d from ICR strain mice were collected, cultured on the fibroblast cell feeder layers that were from the blastocysts of 13.5d from ICR strain mice.The embryonic stem cell colonies were identified, the results can be shown: ① They all have typical morphological characteristics of ES cells, such as the nuclear big, cytoplasm little, high ratio of the nuclear-cytoplasm, the obvious nucleoli, the cells compacted to from colonies, the colonies having strong third dimension, round or elliptic, with smooth edges, with strong refraction, and having no clear boundaries of cells within a colony. ② Positive AKP staining is the typical characteristics of murine undifferentiated ES cells. We checked the activity of AKP of the ES cells using the undifferentiated F0 ES cells from the 3.5d blastocysts which were widely used all over the world as the positive control, and the embryonic fibroblast cells as the negative control. After the AKP staining, all of the ES cells showed strong AKP activity the same as the F0 ES cells.On the contrary, the embryonic fibroblast cells have no expression of AKP showing the lost of staining. ③ One of the criterions for checking the ES cell lines established is the karyotype bearing. A good ES cell line should have at least more 75% cells bearing normal diploid karyotype after 5~10 passages. According to above requirement, there is the ES cells from ICR strain mice with more than 75% normal karyotype. ④ F2 ES cells would differentiated to other cells in the MEF layer without mLIF. In the MEF layer and with mLIF,when the ES cells were cultured to F8 or F11 they would differentiated to other cells too.更多还原