人孤雌胚胎干细胞无饲养层培养体系的建立  

ESTABLISHMENT OF FEEDER-FREE CULTURE SYSTEM OF HUMAN PARTHENOGENETIC EMBRYONIC STEM CELLS

作　　者：梁锐[1] 王志强[2] 陈天星[1] 朱静[1] 朱姝[3] 李英[1] 杨龙[1] 朱宝生[3] 

机构地区：[1]云南省第一人民医院病理科,昆明650032 [2]昆明医科大学第一附属医院肿瘤科 [3]云南省第一人民医院遗传诊断中心,昆明650032

出　　处：《中国修复重建外科杂志》2013年第5期559-564,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：云南省应用基础研究计划项目(2013FZ179)~~

摘　　要：目的建立一种安全、有效、经济且适于人孤雌胚胎干细胞(human parthenogenetic embryonic stemcells,hPESCs)体外培养的无饲养层培养体系。方法将常规体外培养的hPESCs分别以mTeSRTMl培养基(对照组)和人包皮成纤维细胞的条件培养基(human foreskin fibroblasts-conditional medium,hFFs-CM)(实验组)扩增培养,倒置显微镜下观察两组无饲养层培养体系下hPESCs的生长状态;采用ALP检测和核型分析研究hPESCs生物学特性;采用RT-PCR检测hPESCs全能性标记物Oct-4的表达情况;通过体外和体内分化实验观察hPESCs向3个胚层分化的潜能。结果两组hPESCs形态规则、不易分化,在形态、扩增速度等方面无明显差异;已成功在体外培养15代,两组均能保持正常女性的二倍体核型46,XX和全能性;RT-PCR检测示两组Oct-4 mRNA均呈阳性表达;体外分化均可形成拟胚体;在裸鼠体内均可形成含有3个胚层组织成分的畸胎瘤。结论 hFFs-CM无饲养层培养体系可长期支持hPESCs的生长并维持其未分化状态,成功建立了一种不仅能维持hPESCs的有效扩增、减少动物源性污染、降低培养成本,还可满足临床大规模应用的hPESCs无饲养层培养体系。Objective To establish a safe, effective, and economic feeder-free culture system which is suitable for the culture of human parthenogenetic embryonic stem cells （hPESCs） in vitro. Methods hPESCs were cultured with mTeSRTM1 medium （control group） and human foreskin fibroblasts-conditional medium （hFFs-CM） （experimental group）. The growth status of hPESCs in both feeder-free culture systems were observed with inverted microscope. Alkaline phosphatase （ALP） analysis and karyotype analysis were used to study the biological characteristics of hPESCs. The expression of hPESCs pluripotent marker Oct-4 was analyzed by RT-PCR. Differentiation experiment in vivo and in vitro was applied to observe the differentiation potential of hPESCs into three germ layers. Results hPESCs had regular morphology with difficulty in differentiation in both culture systems. No obvious difference was observed in morphology and expansion speed of hPESCs between 2 groups. After subcultured for 15 passages in vitro, hPESCs in 2 groups could maintain normal female diploid karyotype 46, XX and pluripotency. The expression of Oct-4 mRNA was positive in 2 groups, hPESCs in 2 groups could form embryonic body in differentiation experiment in vitro and could develop into teratomas containing three germ layers in nude mice. Conclusion Feeder-free culture system of hFFs-CM can sustain the growth of hPESCs and keep hPESCs undifferentiated state for long. A feeder-free culture system ofhPESCs is successfully established, which can support the growth of hPESCs, reduce the contamination from animals, decrease the cost of culture, and satisfy the clinical laree-scale application.

关 键 词：人孤雌胚胎干细胞 无饲养层 条件培养基 人包皮成纤维细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]