摘要
目的 研究视黄酸对人胚胎干细胞(ESCs)的增殖、干性及其向拟胚体分化的能力的影响,探索应用人ESC为模型研究先天发育畸形机制的可行性.方法 用real-time PCR、MTS细胞增殖分析和免疫荧光染色,鉴定视黄酸处理前、后H9 ESC增殖和干性的变化;用real-time PCR比较H9 ESC向拟胚体分化后,视黄酸处理对三胚层标志基因及成骨和成脂相关基因表达的影响.结果 随着视黄酸处理时间和浓度的增加,H9 ESC的增殖速度在指数生长早期显著上调,干性标志基因OCT4,Nanog和Sox2及Oct4翻译调控因子Lin28的mRNA表达水平显著下降,且Oct4蛋白表达水平也明显降低.视黄酸处理后拟胚体出现空泡结构,外胚层标志基因NeuroD1、Noggin,中胚层标志基因Brachyury、Twist和内胚层标志基因AFP,GATA-4的表达显著上调(P<0.05),并以AFP的变化最为显著(P<0.01).此外,成脂相关基因C/EB Pα表达水平也显著提高,但成骨相关基因OPN的表达水平在视黄酸处理5d后显著下调(P<0.05).结论 高浓度的视黄酸可诱导H9 ESC丧失干性,并使其向拟胚体方向发生过度分化,同时破坏早期拟胚体形成过程中成骨成脂分化的平衡,这可能与其诱发先天性颅面发育畸形相关.
Objective To analyze the influence of retinoic acid (RA) on the undifferentiated state and EB formation abilities of human embryonic stem cells. Methods The biological characteristics of H9 ESCs after RA treatment were characterized by real-time PCR, MTS proliferation assay and immunofluorescence staining. The expression of three germ layers markers, osteogenic differentiation markers and adipogenic differentiation markers in H9-differentiated embryoid bodies (EBs) with RA treatment were quantified by real time PCR. Results The proliferation of H9 ESCs in the early logarithmic growth phase was accelerated by RA treatment. In addition, RA induced differentiation of H9 ESC coupled with morphology changes, decreased expression of undifferentiated markers Oct4, Nanog, Sox2 and OCT4 mRNA binding protein Lin28 at mRNA level, and reduced expression of Oct4 at protein level. RA induced formation of cavities in EBs. Real time PCR results showed that the expressions of ectodermal markers: NeuroD1, Noggin; mesodermal markers: Brachyury, Twist and endodermal markers: AFP, GATA-4 were significantly increased ( P 〈 0.05 ) , especially for AFP ( P 〈 0.01 ), by RA treatment in a dose-dependent manner. In addition, the expression of adipogenic differentiation marker C/EBPα was increased while the osteogenic differentiation marker OPN was decreased in EBs after RA treatment for 5 days. Conclusions High concentrations of RA induced the loss of sternness in H9 ESCs and excessive differentiation in EBs, and damaged the balance between osteogenic and adipogenic differentiation during early EB differentiation, which may be relevant to the congenital malformations.
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2014年第3期191-196,共6页
Chinese Journal of Plastic Surgery
基金
国家自然科学基金(31201102,31071305,30871433)
国家国际科技合作专项项目(2011DFA32190)
关键词
人胚胎干细胞
视黄酸
拟胚体
胚胎发育
Human embryonic stem cells
Retinoic acid
Embryoid body
Embryonic