摘要
目的:研究孤雌胚胎干细胞(phESC)与受精卵来源胚胎干细胞(hESC)在印迹基因表达、X染色体失活等方面的异同。方法:运用实时荧光相对定量PCR、甲基化特异性PCR和免疫荧光染色等方法检测phESC与hESC在父系印迹基因IGF2R,母系印迹基因SNRPN,IGF2相对表达量及X染色体失活状态。结果:①母系印迹基因SNRPN,IGF2在phESC细胞中不表达,而父系印迹基因IGF2R表达量则相对于hESC有近2倍的上调;②XIST基因在第35代phESC细胞中没有表达,意味着早期的phESC没有进行X染色体失活,而到了第55代,XIST基因开始表达并随着分化时间的延长表达量逐渐上调;③XIST启动子甲基化状态及组蛋白H3赖氨酸27三甲基化免疫荧光染色阳性证实phESC在长期培养后启动了X染色体失活。结论:phESC的X染色体失活状态在培养过程中存在不稳定的情况,建议对phESC进行更深入的表观遗传稳定性研究,以确保这种细胞未来安全、高效的应用。
Objective：To investigate the difference of X chromosome inactivation and imprinted gene expression status between the parthenogenetic human embryonic stem cells （phESC） and hESC derived from fertilized embryos. Methods： ①Relative quantitative real-time PCR was used to detect the paternally imprinted gene IGF2R and maternally imprinted gene SNRPN, IGF2 expression levels ② XIST methylation and histone H3 lysine 27 trimethylation （H3K27me3） were used to detected the X chromosome inactivation （XCI） status between the phESC and hESC. Results： ①maternally imprinted gene SNRPN, IGF2 were not expressed in phESC while the paternally imprinted gene IGF2R was expressed with more than 2 fold levels compared to the normal hESCs. ② No expression of XIST gene was detected at passage 35, which indicated that XCI was not initiated in phESC at early passages. After long-term culture, epigenetic status of these cells changed, XIST gene expression could be detected at passage 55 and the expression level was increased after differentiation. ③ DNA methylation status of promoter of XIST and positive H3K27me3 results also confirmed that phESC was initiated XCI. Conclusion：Variation XCI status suggested further investigation is necessary to clarify the epigenetic in phESC in order to ensure the safety of those cells for regenerative medicine.
出处
《现代生物医学进展》
CAS
2010年第3期439-442,F0003,共5页
Progress in Modern Biomedicine
基金
国家自然基金项目(项目编号:30871378)
关键词
孤雌
印迹
X染色体失活
稳定性
Parthenogenetic
Imprint
X chromosome inactivation
Variation
