绵羊类胚胎干细胞的分离与克隆  

The isolation and cloning of sheep embryonic stem-like cells

作　　者：任卫青[1] 陈亮[1] 和小娥[1] 何俊丹[1] 王新庄[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450001

出　　处：《中国兽医学报》2012年第5期788-792,共5页Chinese Journal of Veterinary Science

基　　金：河南省基础与前沿研究计划资助项目(092300410081)

摘　　要：从胚胎发育阶段、饲养层和培养体系等方面对影响绵羊类ES细胞分离、克隆效率的因素进行探讨。结果显示:致密桑葚胚和囊胚的ICM增殖率高于囊胚和孵化囊胚。绵羊类ES细胞在同源绵羊胎儿成纤维细胞(SEF)上生长比较缓慢,最终传代次数也低于小鼠胎儿成纤维细胞(MEF)组。培养液中同时添加胎牛血清(FBS)和Knock-out血清替代品(KSR),绵羊类ES传至7代,添加了碱性成纤维细胞生长因子(bFGF)后,最高可传至8代,而单纯添加KSR或FBS,分别传至4代和5代。对类ES细胞进行AKP染色、核型分析、体外分化试验,证实分离的类ES细胞符合ES细胞的主要特征,而且表达多潜能性细胞因子Nanog。由此认为,致密桑葚胚和囊胚更适合绵羊类ES细胞的体外分离和培养,而且MEF更适合于绵羊类ES细胞的分离传代,培养液中添加5%FBS和15%KSR,比较适合类ES细胞的分离传代,bFGF对绵羊类ES细胞的增殖具有促进作用。Experiment were performed to determine the effect factor of solation,cloning and passage of embryonic stem cells（ES）in sheep,including developmental stage of embryos,feeder cells and growth factor.The result showed that The ICM growing rate of compacted morula and blastocysts were better than that of morula and hatching embryos.The growth rate and the capability of maintained undifferentiation of sheep ES-like cells which were cultured on MEF,are better than those on SEF.We applied basic medium with 15% KSR and 5% FBS as a new culture system to isolate sheep ES-like cells,the ES-like cell line maintained undifferentiating for7 passages.Added bFGF at the same time,it maintained undifferentiating for 8 passages,but when applied only 15% KSR or 15% FBS,it only could remain undifferentiated for 4 passages or 5 passages.Sheep ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase.Multiple differentiation potency of the ES-like cells was confirmed by expressing a novel ES cells specifically expressed transcription factor（Nanog）and forming embryoid bodies in suspension culture.These results suggest that compacted morula and blastocysts are the suitable material for the isolation and cloning of sheep ES-like cells,and The MEF is more suitable for the isolation and passage of sheep ES-like cells.Additionally,the results also indicated that both the 15% KSR and 5% FBS which were supplied in the culture system are suitable for cloning of sheep ES-like cells,and bFGF can promote sheep ES-like cells＇ self-renewal.

关 键 词：绵羊 胚胎干细胞 分离与培养 饲养层 克隆 

分 类 号：S826.3[农业科学—畜牧学]