免疫外科法分离克隆BALB/c小鼠胚胎干细胞  

Isolation and Culture of Embryonic Stem Cells from BALB/c Strain Mouse by Immunosurgery

作　　者：徐小明[1] 窦忠英[1] 华进联[1] 葛秀国[1] 

机构地区：[1]西北农林科技大学陕西省干细胞工程技术研究中心,杨凌712100

出　　处：《中国实验动物学报》2004年第3期129-133,i001,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金项目 (3 0 0 70 3 74);陕西省重点科技计划项目资助。

摘　　要：目的　使用免疫外科法分离克隆BALB c小鼠胚胎干细胞 (embryonicstemcells,ES细胞 ) ,为进一步建立BALB c小鼠ES细胞系打下基础。方法　用免疫外科法从 4 5枚BALB c小鼠囊胚中分离得到 2 0枚去除滋养层细胞的ICM ,接种在MEF饲养层上 ,使用DMEM(高糖 ) +15 %FBS +0 1mmol Lβ 巯基乙醇 +0 0 1mmol L非必需氨基酸 +10 0 0IU mlLIF +10 0IU mL青霉素 +10 0IU ml链霉素培养液 ,17枚形成典型的ICM集落 (85 0 % ) ,有一枚胚胎传至第 10代。用于全胚培养的BALB c小鼠胚胎共 10 2枚 ,使用与免疫外科相同的培养方法 ,6 6枚形成典型的ICM集落 (6 4 7% ) ,其中一枚胚胎传至第 8代。结果　免疫外科法较全胚培养法有利于小鼠ES细胞的分离与克隆 ;添加LIF(10 0 0IU ml)有利于小鼠ES细胞的分离与传代 (P <0 0 5 ) ;0 0 5 %胰酶 +0 0 0 8%EDTA是较好的ES细胞消化液 ,对细胞综合损伤力小 ,且传代后ES细胞集落形成能力也较高 (P <0 0 5 )。结论　分离得到的ES细胞经形态学观察 ,AKP染色 ,体外分化实验 ,核型分析等证明其具有胚胎干细胞的诸多特性。Objective To isolate and culture mouse embryonic stem (ES) cells using immunosurgical method, which would lay the foundations of further establishing BALB/c strain mouse ES lines. Methods 20 pure ICMs were obtained from 45 embryos of BALB/c strain mouse using immunosurgical method. Then ICMs were inoculated on mitomycin-inactivated MEF feeders. ES-like colonies were observed from 17 of 20 pure ICMs and one ES-like cell line had maintained undifferentiation for 10 passages. In addition,ES-like colonies were also obtained from 66 of 102 embryos of BALB/c strain mouse using a method of intact embryo culture. Both methods used mouse ES culture medium composed of DMEM (high glucose) containing 15% FBS, 0.1 mmol/L β-mercaptoethanol, 0.01 mmol/L non-essential amino acid, 1000 IU/ml LIF, 100 IU/ml penicillin, and 100 IU/ml streptomycin. Results Immunosurgical method was better than intact embryo cultural method in the isolation and culture of mouse ES cells. Supplement with LIF (1000 IU/ml) is beneficial to the isolation and culture of mouse ES cells (P<0.05); 0.05% trypsin-0.008% EDTA is a good digest for mouse ES cells and is better for forming new ES colonies (P<0.05). Conclusions The mouse ES cells which are pluripotential cells have been identified by colony morphology, AKP staining, in vitro differentiation and karyotype analysis.

关 键 词：BALB/C小鼠 胚胎干细胞 免疫外科法 分离 克隆 

分 类 号：Q813[生物学—生物工程] Q785