无血清分离昆明系小鼠胚胎干细胞  

Isolation and culture of embryonic stem cells from mouse embryos of Kunming species in the serum-free condition

作　　者：余树民[1] 严兴荣[1] 陈冬梅[1] 王晗[1] 窦忠英[1] 

机构地区：[1]西北农林科技大学动物科技学院国家干细胞工程技术研究中心陕西分中心

出　　处：《中国兽医学报》2008年第3期328-332,共5页Chinese Journal of Veterinary Science

基　　金：国家“863”计划资助项目(2005AA21905);教育部重大科技资助项目(03160);陕西省重大科技专项资助项目(2006KZ05-G1)

摘　　要：以昆明系小鼠胚胎为研究材料,以丝裂霉素C处理的胎鼠成纤维细胞（Mouse embryonic fibroblasts,MEF）为饲养层,在胚胎干细胞（Embryonic stemcells,ESCs）培养液中添加血清替代物（Knockout serumreplacement,KSR）以替代胎牛血清（Fetal bovine serum,FBS）,并与添加FBS的培养液进行对比,研究了昆明系小鼠胚胎贴壁、ICM（Inner cell mass,ICM）集落形成以及ESCs分离的情况。结果表明,在添加KSR的培养液中,胚胎在培养3~5 d贴壁,胚胎贴壁率显著低于添加FBS的培养液;5~7 d形成ICM集落,集落未分化形态可维持2~3 d,培养10 d ICM集落仍可保持典型的未分化形态;ICM集落形成率和1代ESCs集落出现率与添加FBS的培养液相比差异不显著（P〉0.05）,但2~5代ESCs集落出现率显著高于添加FBS的培养液,其中有2株ESCs在添加KSR的培养液中传到7代;所分离细胞显示碱性磷酸酶染色强阳性,Oct-4、Nanog的免疫组化染色阳性,具有ESCs的特点。结论：在ESCs培养液中添加KSR比添加FBS更有利于维持ICM集落及ESCs的未分化状态。The experiment investigated embryo attachment,formation of ICM outgrowths and isolation of embryonic stem cells（ESCs） from mouse embryos of Kunming specie which were used as the starting material while mitomycin C-inactivated mouse embryonic fibroblasts as the feeder layer,in the medium supplemented with Knockout serum replacement（KSR） instead of fetal bovine serum（FBS） as compared with FBS.The results demonstrated that in the mediun supplemented with KSR,most of embryos attached to the dish with the 5-6 days cultured,and frequencies of embryo attachment was significantly lower than that in the medium supplemented with FBS（P〈0.05）.Meanwhile,inner cell mass（ICM） outgrowths appeared in the 5-7 days and their undifferentiated morphologies sustained 2-3 days until more or less the tenth day cultured,then formation rates of ICM outgrowths and ESCs clonies at the first passage existed no significant difference as compared with that in the medium supplemented with FBS（P〉0.05）,however the formation rates of ESCs clonies at the 2-5 th passages were higher than those in the medium supplemented with FBS（P〈0.05）,two cell lines among the isolated cells were passaged to the seventh passages.The isolated cell lines,which were positive for alkaline phosphatase staining and also immunohistiochemically positive against antigens of Oct-4 and Nanog,had the similar antigen pattern to ESCs.In conclusion,it was advantageous to sustain the undifferentiated status of ICM outgrowths and ESCs clonies in the ESCs medium supplemented with KSR over FBS.

关 键 词：昆明系小鼠 胚胎 胚胎干细胞(ESCs) 内细胞团(ICM) 

分 类 号：S814.8[农业科学—畜牧学] S865.13[农业科学—畜牧兽医]