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12.5～14.5d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3.5d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。
Objective To 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 by alkaline 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 a colony-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.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2012年第6期1081-1085,I0011,I0012,共7页
Journal of Jilin University：Medicine Edition
基金
国家重点基础研究发展计划(973计划)项目资助课题(2011CB707902)
