摘要
以荷斯坦牛胚胎和小鼠胚胎为材料 ,研究了犊牛血清、饲养层、培养液、添加物和消化液对牛胚胎干细胞和小鼠胚胎干细胞克隆效率的影响。结果表明 ,在 2 4h内使小鼠胚胎贴壁率达 86 %以上的犊牛血清可用于小鼠和牛胚胎干细胞的分离 ;在 ES细胞分离与克隆中 ,以 15 %～ 2 0 %犊牛血清为宜 ,在 DMEM(L)培养基中添加 0 .1μmol/LNa2 Se O3 +0 .1mmol/Lβ-巯基乙醇 +10 μg/L IGF+10 0 0 IU/m L L IF,能显著提高牛 ES细胞分离与克隆效率 ;在TCM199、DMEM(高糖 )和 DMEM(低糖 ) 3种培养基中 ,低糖 DMEM更适宜于牛 ES细胞的分离 ;优秀胚胎形成的团状 ICM更适宜于分离与克隆 ES细胞 ,在 37℃用低浓度消化液处理 ICM或 ES细胞集落 ,再以机械将其离散为细胞小块 ,ES细胞克隆效率最高。
The effect of some factors such as calve serum,feeders,culture medium,additives on efficiency of isolation and cloning of bovine embryonic stem cells(ESC) were studied with bovine embryos and murine embryos.The results showed that:firstly,the high efficiency of islolation and cloning of bovine ES cells were obtained by supplementing with 15% super NBS(the rate of mouse embryo hatched was more than 86% by 24 h of culture);secondly,the rate of bovine ES colonies cultured in DMEM(low glucose) was higher than those cultured in DMEM(high glucose) and TCM199;thirdly,super bovine embryos formed mass ICM,the efficience of ES cell clone was higher than stream ICM and net ICM derived nomal embryos;fourthly,the rate of cloning of bovine ES cell was the highest in DMEM(low glucose) culture medium supplemented with 15% NBS,0.1 μmol/L Na 2SeO 3,0.1 mmol/L β-mercaptoethanol(Sigma), 1 000 IU/mL LIF and 10 μg/L IGF;at last,when ICM or ES cell colonies were treated with 0.05% trypsin and 0.01% EDTA for long term(6 min),the rate of cloning of bovine ES cell was the highest in this research.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2002年第2期187-190,共4页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目 ( 396 70 35 9)
国家攻关课题 ( 96 -C0 10 3)
关键词
牛
小鼠
胚胎干细胞
内细胞团
分离
克隆
bovine
murine
embryonic stem cell
inner mass
isolation
clone
