中文摘要：

以昆明系小鼠（Mus musculus Km）胚胎为材料,以丝裂霉素（10μg/mL）处理的MEF为饲养层,研究了在胚胎干细胞（embryonic stem cells,ESCs）培养液中分别添加血清替代物（knockout serum replacement,KSR）、胎牛血清（fetal bovine serum,FBS）和FBS＋PD98059（50μmol/L）对昆明系小鼠胚胎贴壁、内细胞团（inner cell mass,ICM）集落形成及ESCs分离培养的影响。结果表明,在ESCs培养液中添加KSR,胚胎贴壁率显著低于添加FBS（P〈0.05）,ICM集落形成率和1代ESCs集落出现率差异不显著（P〉0.05）,2-5代ESCs集落出现率显著高于添加FBS（P〈0.05）,2株ESCs传到7代;在ESCs培养液中添加FBS＋PD98059,胚胎贴壁率、ICM集落形成率和1-5代ESCs集落出现率均显著低于添加KSR或FBS（P〈0.05）;用0.5g/L胰酶＋0.2g/L EDTA离散消化ICM细胞和ESCs并结合机械分割,1-5代ESCs集落出现率显著高于用2.5g/L胰酶＋0.2g/LEDTA（P〈0.05）。实验结果表明,在ESCs培养液中添加KSR,较添加FBS或FBS＋PD98059更适合用于分离培养昆明系mESCs,用0.5g/L胰酶＋0.2g/L EDTA离散消化ICM细胞和ESCs并结合机械分割优于用2.5g/L胰酶＋0.2g/L EDTA。

英文摘要：

This experiment focused on investigating effects of different supplements in the media containing knockout serum replacement （KSR）, fetal bovine serum （FBS） and FBS＋PD98059 （50 μmol/L）, respectively on embryo attachment, formation of inner cell mass （ICM） outgrowths, isolation and culture of embryonic stem cells （ESCs） from mouse （Mus musculus Km） embryos of Kunming species , which was used as the experimental material while mitomycin C-inactivated mouse embryonic fibroblasts （MEF） as the feeder layers. The results demonstrated that the efficiencies of embryo attachment in the medium supplemented with KSR was significantly lower compared with that of the medium supplemented with FBS（P 〈 0.05）, and formation efficiencies of ICM outgrowths and ESCs clonies at the first passage appeared no significant difference between them （P 〉 0.05）, however, formation efficiencies of ESCs clonies at the 2-5 passages were significantly higher than those in the medium supplemented with FBS （P 〈 0.05）, two ESCs lines were subcultured to the seventh passages. Meanwhile, the efficiencies of embryo attachment, formation of ICM outgrowths and ESCs clonies at the 1-5 passages in the medium supplemented with FBS ＋ PD98058 （50 μmol/L） were significantly lower than those in the medium supplemented with KSR or FBS, respectively （P 〈 0.05）. Providing ICM cells and ESCs were trypsinized with 0.5 g/L trypsin ＋ 0.2 g/L EDTA combined with manual dissection, ESCs clonies at the 1-5 passages appeared significantly higher than those with 2.5 g/L trypsin ＋ 0.2 g/L EDTA （P 〈 0.05）. Therefore, the results indicated that it was advantageous to isolation and culture of ESCs from mouse embryos of Kunming Species in the medium supplemented with KSR over FBS and FBS＋PD98059 （50 μmol/L）, respectively. Accordingly, the ICM cells and ESCs at the initial passages were trypsinized more properly with 0.5 g/L trypsin associated with manual dissociation, than with 2.5 g/L trypsin.