人孤雌胚胎干细胞无饲养层培养体系的建立

发布时间：2018-01-18 08:02

本文关键词：人孤雌胚胎干细胞无饲养层培养体系的建立　出处：《中国修复重建外科杂志》2013年05期 　论文类型：期刊论文

【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。
[Abstract]:Objective to establish a safe, effective, economical and suitable human parthenogenetic embryonic stemcells for human parthenogenetic embryonic stem cells. Methods hPESCs cultured in vitro was cultured in mTeSRTMl medium (control group). And human foreskin fibroblasts-conditional medium. The growth status of hPESCs was observed under inverted microscope under the culture system without feeding layer. ALP detection and karyotype analysis were used to study the biological characteristics of hPESCs. The expression of Oct-4, a potent marker of hPESCs, was detected by RT-PCR. The differentiation potential of hPESCs into three embryo layers was observed in vitro and in vivo. Results the morphology of hPESCs in the two groups was regular and it was not easy to differentiate. There was no significant difference in morphology and expansion speed between the two groups. After 15 generations of culture in vitro, both groups were able to maintain the diploid karyotype 46X and totipotency of normal women. RT-PCR showed that Oct-4 mRNA was positive in both groups. In vitro differentiation can form embryoid body. Teratoma containing three tissue components of the embryo layer could be formed in nude mice. Conclusion hFFs-CM culture system without feeder layer can support the growth of hPESCs for a long time and maintain its undifferentiated state. A culture system without feeding layer for hPESCs was successfully established, which can not only maintain the effective amplification of hPESCs, reduce animal pollution and reduce the cost of culture, but also meet the needs of large scale clinical application of hPESCs.
【作者单位】：云南省第一人民医院病理科;昆明医科大学第一附属医院肿瘤科;云南省第一人民医院遗传诊断中心;
【基金】：云南省应用基础研究计划项目(2013FZ179)~~
【分类号】：R329
【正文快照】： 人孤雌胚胎干细胞(human parthenogenetic embryonicnet/kcms/detail/51.1372.R.20130423.1840.006.htmlstem cells,hPESCs)是通过人工方法(不经过精子受精)激活卵母细胞获得人孤雌囊胚,再分离其中的内细胞团所得的人胚胎干细胞(human embryonic stemcells,hESCs)。它与hESCs

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