添加物和消化液对牛和小鼠类胚胎干细胞克隆效率的影响  

The influence of supplement and digestant on isolation and clone of bovine and murine embryonic stem like cells

作　　者：安立龙[1] 高志敏[1] 杨奇[1] 冯秀亮[1] 窦忠英[1] 雷安民[1] 杨春荣[1] 邱怀[1] 

机构地区：[1]西北农林科技大学家畜生殖内分泌与胚胎工程农业部重点开放实验室

出　　处：《西北农林科技大学学报（自然科学版）》2001年第5期5-10,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金资助项目 ( 396 70 35 9) ;国家"九五"攻关项目 ( 96 -C0 10 3) ;西北农林科技大学青年教师专项基金资助项目 ( 5 2 10 818)

摘　　要：研究了犊牛血清、饲养层、培养液、添加物和消化液对牛和小鼠类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和克隆 ;在 ES细胞培养液中 ,犊牛血清体积分数以 15 %～ 2 0 %为宜 ,在 DMEM(L )培养液中添加 0 .1μm ol/ L Na2 Se O3+0 .1mm ol/ Lβ-巯基乙醇 +10 ng/ m L IGF+10 0 0 ng/ m L L IF,能显著提高牛 ES细胞分离与克隆效率 ;低糖 DMEM比TCM199和高糖 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℃用低体积分数消化液处理 ICM或 ES细胞集落 ,再以机械将其离散为细胞小块 。The effect of new bovine serum,feeders,culture medium,additives and digestive juice on efficiency of isolation and clone of bovine embryonic stem cells (ESC) was studied through bovine embryos and murine embryos.The results showed:First,the high efficiency of isolation and clone of bovine ES cell was obtained by supplementing with 15% super NBS (the ration of mouse embryo hatched are more than 86% within 24 h culture).Second,the ration of bovine ES colonies cultured in DMEM(low glucose) were more than those cultured in DMEM (high glucose) and TCM199.Third,super Bovine embryos formed mass ICM,the efficiency of ES cell clone derived mass ICM was higher than that of stream ICM and net ICM derived normal embryos.Forth,the ration of clone of bovine ES cell was highest in DMEM (low glucose) culture medium supplemented with 15% NBS,0.1 μmoL/L Na 2SeO 3,0.1 moL/L β mercaptoethanol (Sigma),1 000 ng/mL LIF and 10 ng/mL IGF.Last,when ICM or ES cell colonies were treated with \{0.05%\} trypsin and 0.01% EDTA for long period (6 min),the efficiency of clone of bovine ES cell was highest in this research.

关 键 词：牛 小鼠 胚胎干细胞 内细胞团 克隆效率 添加物 消化液 

分 类 号：S823.36[农业科学—畜牧学] Q954.4[农业科学—畜牧兽医]