小鼠成纤维细胞饲养层的制备及C57BL/6小鼠胚胎干细胞系的建立

J4 ›› 2012, Vol. 38 ›› Issue (6): 1081-1085.

小鼠成纤维细胞饲养层的制备及C57BL/6小鼠胚胎干细胞系的建立

张小梅|方廖琼|王智彪

（重庆医科大学生物医学工程学院　省部共建超声医学工程国家重点实验室 超声医学工程重庆市市级重点实验室|重庆 400016）

收稿日期:2012-04-28 出版日期:2012-11-28 发布日期:2012-12-28
通讯作者: 王智彪 E-mail:（Tel：023-68485021，E-mail:wangzhibiao@haifu.com）
作者简介:张小梅(1986-)|女|重庆市人|医学硕士|主要从事胚胎干细胞与肿瘤相互作用关系方面的研究。
基金资助:
国家重点基础研究发展计划（973计划）项目资助课题（2011CB707902）

Preparation of mouse fibroblast feeder layers and establishment of embryonic stem cell lines of C57BL/6 mice

ZHANG Xiao-mei,FANG Liao-qiong,WANG Zhi-biao

(Key Laboratory of Ultrasound Engineering in Medicine Co-founded by Chongqing and Ministry of Science and Technology,Chongqing Key Laboratory of Ultrasound Engineering in |Medicine,School of Biomedical Engineering,Chongqing Medical University,Chongqing 400016,China）

Received:2012-04-28 Online:2012-11-28 Published:2012-12-28

摘要/Abstract

摘要：

目的：制备小鼠成纤维细胞饲养层并建立C57BL/6小鼠胚胎干细胞（ESC）系，为进一步建立人ESC系提供依据。方法：取妊娠12.5~14.5 d的胎鼠，组织消化法分离培养小鼠胚胎成纤维细胞（MEF）；收集C57BL/6小鼠3.5 d的囊胚培养于小鼠制作的饲养层上；分离内细胞团(ICM)，扩增传代40代以上。观察ESC集落的生长情况。采用碱性磷酸酶(AKP)染色、免疫组织化学早期胚胎特异性表面抗原(SSEA-1)、八聚体结合转录因子4(OCT-4)染色和体内分化实验对ESC集落进行鉴定。结果：分离培养后得到的ESC可稳定传代至40代以上，且均呈集落样生长。经AKP、SSEA-1和OCT-4染色后ESC均呈红褐色阳性表达，接种于裸鼠均可形成畸胎瘤；HE染色，有3个胚层组织成分。结论：成功建立了C57BL/6小鼠ESC系。

关键词: 胚胎成纤维细胞；C57BL/6小鼠；胚胎干细胞；碱性磷酸酶

Abstract:

o prepare mouse fibroblast feeder layers and to establish C57BL/ 6 mouse embryonic stem cell(ESC) lines and to provide basis for the further establishment of human ESC lines.Methods The mouse embryonic fibroblasts(MEF) were isolated from the mouse embryos at 13.5-14.5 d by primary tissue digestion method and the 3.5 d blastulae of C57BL/6 mice were cultured on the mouse fibroblast feeder layer incubation;the inner cell mass(ICM) was separated and amplified and passaged over 40 generations.The growth of the MEF colonies was observed.The ESC colonies were identified byalkaline phosphatase(AKP) staining,early embryo-specific surface antigen(SSEA-1) and octamer-blinding transcription factor 4(OCT-4) staining,and differentiation experiments in vivo.Results The ESC after isolation and culture could stably passage to more than 40 generations and showed acolony-like growth.The ESC showed a red-brown positive expression after AKP, SSEA-1 and OCT-4 staining.The teratomas formed after ESC was inoculated in nude mice.HE staining showed that there were three germ layer tissue components in ESC.Conclusion The C57BL/6 mouse ESC lines are successfully established.

Key words: embryonic fibroblast;C57BL/6 mouse;embryonic stem cell;alkaline phosphatase

中图分类号:

Q132.8

张小梅|方廖琼|王智彪. 小鼠成纤维细胞饲养层的制备及C57BL/6小鼠胚胎干细胞系的建立[J]. J4, 2012, 38(6): 1081-1085.

ZHANG Xiao-mei,FANG Liao-qiong,WANG Zhi-biao. Preparation of mouse fibroblast feeder layers and establishment of embryonic stem cell lines of C57BL/6 mice[J]. J4, 2012, 38(6): 1081-1085.