上皮间质转化(epithelial-mesenchymal transition,EMT)是指上皮细胞失去连接和极性转变为间质细胞的过程,这一现象普遍存在于胚胎发育、创伤愈合、器官纤维化以及肿瘤转移.在胚胎早期发育和晚期发育过程,例如着床、原肠运动、心血管发育等事件中有EMT和间质上皮转化(mesenchymal-epithelial transition,MET)的参与.EMT和MET参与调控干细胞表型变化、细胞迁移运动,是细胞差异分化和三维组织构建的重要机制.EMT的重要标志是细胞黏附分子表达由E-钙黏着蛋白(E-cadherin)向N-钙黏着蛋白(N-cadherin)转换.E-钙黏着蛋白通过与β-联蛋白、p120-联蛋白、a-联蛋白联合,影响Wnt、小GTP酶超家族等信号通路活化,调控细胞骨架运动.TGFβ、Notch、Wnt、BMP、FGF等信号通路,Snail、Twist、Zeb等转录因子,联合表观修饰酶,协同参与EMT的启动和调控.体外研究模型表明,E-钙黏着蛋白参与干细胞自我更新;而体细胞重编程可视为MET,重编程因子辅助体细胞获得E-钙黏着蛋白表达.体外研究发现,EMT及相关分子(例如E-钙黏着蛋白、Snail、Twist、Zeb等)参与了早期三胚层分化及晚期特定细胞类型的形成.对EMT机制的研究有助于理解和改善干细胞体外诱导分化效率,促进类器官的构建和诱导.

目的:探讨程序性细胞死亡蛋白4(programmed cell death protein 4,PDCD4)对人类慢性粒细胞白血病细胞系K562细胞活力、凋亡以及基因表达变化的影响.方法:通过慢病毒感染的方法在K562细胞系中过表达PDCD4基因并筛选获得稳定表达的细胞系;采用RT-qPCR和Western blot分别检测PDCD4 mRNA和蛋白的表达水平;CCK-8法检测细胞活力;流式细胞术检测细胞周期和细胞凋亡情况;应用转录组测序(RNA sequencing,RNA-seq)分析PDCD4过表达组和对照组的基因表达差异及其相关的功能与信号通路,RT-qPCR验证测序结果的准确性.结果:成功建立了稳定过表达PDCD4的K562细胞系;PDCD4过表达可显著抑制K562细胞的活力,引起细胞周期阻滞并促进阿糖胞苷诱导的细胞凋亡(P<0.05);RNA-seq结果显示,在K562细胞中过表达PDCD4可引起大量的基因表达发生变化,表达明显差异的基因共有394个,其中上调16个,下调378个,主要涉及细胞周期、凋亡、细胞自噬和代谢等方面,选取的6个基因的RT-qPCR检测结果和RNA-seq的结果一致.结论:PDCD4可抑制K562细胞活力、阻滞细胞周期进展、促进细胞凋亡;RNA-seq确定了PDCD4对K562细胞基因表达谱的变化.

4-phenylbutyrate (4-PBA), a terminal aromatic substituted fatty acid, is used widely to specifically attenuate endoplasmic reticulum (ER) stress and inhibit histone deacetylases (HDACs). In this study, we investigated the effect of 4-PBA on cardiac differentiation of mouse embryonic stem (ES) cells. Herein, we found that 4-PBA regulated cardiac differentiation in a stage-specific manner just like trichostatin A (TSA), a well-known HDAC inhibitor. 4-PBA and TSA favored the early-stage differentiation, but inhibited the late-stage cardiac differentiation via acetylation. Mechanistic studies suggested that HDACs exhibited a temporal expression profiling during cardiomyogenesis. Hdac1 expression underwent a decrease at the early stage, while was upregulated at the late stage of cardiac induction. During the early stage of cardiac differentiation, acetylation favored the induction of Isl1 and Nkx2.5, two transcription factors of cardiac progenitors. During the late stage, histone acetylation induced by 4-PBA or TSA interrupted the gene silence of Oct4, a key determinant of self-renewal and pluripotency. Thereby, 4-PBA and TSA at the late stage hindered the exit from pluripotency, and attenuated the expression of cardiac-specific contractile proteins. Overexpression of HDAC1 and p300 exerted different effects at the distinct stages of cardiac induction. Collectively, our study shows that timely manipulation of HDACs exhibits distinct effects on cardiac differentiation. And the context-dependent effects of HDAC inhibitors depend on cell differentiation states marked by the temporal expression of pluripotency-associated genes.

表观遗传调控,如组蛋白乙酰化修饰,是决定干细胞分化方向的重要机制.组蛋白去乙酰化酶抑制剂(HDACi)通过影响不同亚类的组蛋白去乙酰化酶(HDAC)活性,提高组蛋白乙酰化水平,调控基因表达,从而影响胚胎干细胞自我更新,以及沿神经元、心肌和造血等细胞谱系的定向分化.HDACi类小分子化合物在体细胞重编程中也有广泛的应用,可替代致癌因子c-Myc和Klf4,促进体细胞克隆.研究显示,HDACi的效应与药物剂量、细胞类型和细胞分化状态密切相关.本文主要阐述了HDACi在干细胞分化和体细胞重编程中的应用进展,并对所涉及的分子通路进行讨论,有助于揭示干细胞定向分化的关键分子机制,优化干细胞定向分化诱导策略,对干细胞诱导分化具有重要的理论和实用价值.

Cardiac differentiation in vitro is a complex, stepwise process that is rigidly governed by a subset of transcription factors and signaling cascades. In this study, we investigated the cooperation of cardiac-specific transcription factors Gata4 and Nkx2.5, as well as mitogen-activated protein kinase (MAPK) cascades. P19 embryonic carcinoma cells were induced into spontaneously beating cardiomyocytes utilizing a two-step protocol that comprised an early stage and a late stage of differentiation. During early-stage differentiation in suspension culture, P19 cells aggregated to form embryoid bodies (EBs), and the Gata4 and Nkx2.5 genes were induced. However, Gata4 expressed at the early stage of differentiation was incapable of activating downstream gene expression, as it was localized in the cytoplasm and prone to degradation. After EBs were plated for late-stage differentiation in adherent culture, the MAPK cascades were highly activated and contributed to the activation of Gata4 and Nkx2.5. Specifically, we revealed that p38 signaling participated in regulating the localization and stabilization of Gata4 and Nkx2.5. Additionally, the JNK cascade regulated late-stage cardiac differentiation; JNK kinase reduced Gata4 stabilization and conversely alleviated Nkx2.5 degradation by direct interaction and phosphorylation of Nkx2.5. Finally, we found that the C-terminal domain of Nkx2.5 was required for its stabilization under conditions of oxidative stress and JNK activation. Overall, our results indicated that the induction and activation of Gata4 and Nkx2.5 during early- and late-stage cardiac differentiation was closely associated with the function of the MAPK signaling cascades.