| 不同添加物对分离昆明系小鼠胚胎干细胞的影响研究 |
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| 引用本文： | 余树民,严兴荣,王,晗,窦忠英.不同添加物对分离昆明系小鼠胚胎干细胞的影响研究[J].农业生物技术学报,2008,16(1). |
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| 作者姓名： | 余树民 严兴荣 王 晗 窦忠英 |
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| 作者单位： | 西北农林科技大学国家干细胞工程技术研究中心陕西分中心。 西北农林科技大学国家干细胞工程技术研究中心陕西分中心 |
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| 摘 要： | 本试验以昆明系小鼠胚胎为材料，以丝裂霉素（10μg/mL）处理的MEF为饲养层，研究了在ESCs培养液中分别添加KSR、FBS、FBS+PD98059 (50 μmol/L)对昆明系小鼠胚胎贴壁、ICM集落形成及ESCs分离培养的影响。结果表明，在添加KSR的ESCs培养液中，胚胎贴壁率显著低于添加FBS的ESCs培养液（P＜0.05），但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的ESCs培养液（P＜0.05），2株ESCs被传到7代；在添加PD98059+FBS的ESCs培养液中，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的ESCs培养液（P＜0.05）；用0.5 g/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2.5 g/L+0.2 g/L EDTA（P＜0.05）。结论：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小鼠ESCs，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2.5 g/L+0.2 g/L EDTA。
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| 关 键 词： | 昆明系小鼠 胚胎 胚胎干细胞（ESCs） 内细胞团（ICM） |
| 收稿时间： | 2007-5-2 |
| 修稿时间： | 2007-7-19 |
Investigation on Affects of Different Supplements in the Medium on Isolation and Culture of Embryonic Stem Cells from Mouse Embryos of Kunming Species |
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| Abstract: | This experiment focued on investigating affects of different supplements in the media containing KSR, FBS, FBS+PD98059, respectively on embryo attachment, formation of ICM outgrowths, isolation and culture of embryonic stem cells (ESCs) from mouse 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 supplied with KSR was significantly lower compared with that of the medium supplied 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, were significantly lower than those in the medium supplemented with KSR and FBS, respectively（P＜0.05). Providing ICM cells and ESCs were trypsinized with 0.5g/L trypsin +0.2g/L EDTA combined with manual dissection, ESCs clonies at the 1~5 passages appeared significantly higher than those with 2.5g/L trypsin + 0.2g/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, 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. |
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