中文摘要：

为了更高效地分离昆明小鼠胚胎干细胞,本研究从饲养层、胚胎发育阶段和培养液方面进行优化。将3代以内的小鼠胎儿成纤维细胞（MEF）用丝裂霉素C处理后,分别按1×104、1×105、1×106·mL-1密度接种,以H-DMEM＋15%KSR＋LIF为培养液,观察不同密度饲养层对昆明小鼠胚胎干细胞（ES细胞）生长的影响,并研究胚胎发育阶段和培养液中分别添加干细胞生长因子（SCF）、SCF＋胰岛素对昆明小鼠ES细胞分离克隆的影响。结果显示,胚胎在密度为1×105·mL-1的饲养层上,F1代和F2代ES细胞克隆形成率均显著高于其他2组（P〈0.05）。囊胚的F2代ES细胞克隆形成率显著高于桑椹胚（P〈0.05）,培养液中添加SCF显著提高昆明小鼠胚胎贴壁率（P〈0.05）,同时添加SCF和胰岛素得到昆明小鼠最高胚胎贴壁率及F1、F2代ES细胞克隆形成率。所分离的ES细胞显示AKP染色强阳性,Oct-4、SSEA-1的免疫荧光检测阳性,具有ES细胞的特点。由此认为,发育至囊胚的胚胎在MEF密度为1×105·mL-1上,培养液中同时添加SCF和胰岛素更适合昆明小鼠ES细胞的分离培养。

英文摘要：

In order to isolate the ES cells from Kunming mouse efficiently, the feeder cells, devel- opmental stage of embryos and culture conditions were optimized in the study. Mouse embryonic fibroblast （MEF） were inoculated at concentrations of 1×10^4、1×10^5、1×10^6·mL^-1 as the feeder layers of ES cells after they were treated with Mitomycin C within 3 passages, and cultured inH-DMEM＋15%KSR＋LIF, the growth of the ES cells were observed. The effects of developmental stages of embryos and added SCF, SCF＋insulin in culture medium on growth of Kunming mouse ES cells were investigated. The results showed that the formation rates of the 1st and 2nd passage ES cells in 1 × 10^5·mL^-1 MEF were higher than those cultured on the other 2 groups （P〈0.05）. The 2nd passage ES cells colonies formation rate of blastocysts were higher than that of compacted morula（P〈0.05）. It had improved the attachment rate was significantly enhanced （P〈0.05） when the culture medium added with SCF, and the highest attachment rate, 1st and 2nd passage ES cells colonies formation rate were got when the culture medium added with SCF and insulin （P〈0.05）. The isolated ES cells, which were positive for AKP staining and immunofluorescence against antigens of Oct-4 and SSEA-1, had a series of characters of mice ES cells. These results suggest that the blastocysts on the MEF with a density of 1 × 10^5·mL^-1 and addition with SCF and insulin in the culture medium is more suitable {or the isolation and passage of Kunming mouse ES cells.