二噁英和维生素A在腭裂诱发中的相互作用及机制

Dioxins are widespread endocrine disruptors, which are classified as a Group 1 human carcinogens and teratogenic agents. Vitamin A is one of the essential nutrients for maintaining the biological function of human body. Previous studies revealed that dioxins induce a broad spectrum of biochemical and toxic effects in mammals including alterations of the vital retinoid (vitamin A) system. In our data of dioxins-induced cleft palate, we found that vitamin A deficiency or excess could elevate the teratogenic effects of dioxins. Evidence from gene knockout models confirmed that key genes involved in vitamin A metabolism might exert functions on dioxin induced cleft palate. Findings from in vitro studies suggest that Oct4 could suppress the effects of vitamin A on the signaling pathways of dioxins. Thus, we hypothesized that there are crosstalk effects between dioxins and vitamin A in the process of dioxins-induced cleft palate, and Oct4 might be one of the key molecules involved in the above process. In order to test this hypothesis, via utilizing multiple cleft palate models in vitro and in vivo, knockout mice models, immunfluorescence histochemical double-staining, in situ hybridization, LacZ tracer, ChIP-qPCR, siRNA techniques, this proposal will explore in the second palate development, the molecular and pathological modulatory roles of Oct4 in the crosstalk between dioxins and vitamin A. We will also explore the role of Oct4 in cell migration, secretion, division and disintegration of the medial epithelial seam during the fusion of the palatine shelves. We believe that clinically our project will lead to more effective treatment of facial clefting.

二噁英是有致畸效应的内分泌干扰物。维生素A是人体必需营养素。研究表明，二噁英能够抑制并消耗维生素A在肝脏中的储存，影响维生素A的代谢及稳态。我们在腭裂诱发实验中发现，维生素A缺乏或过量均能提高二噁英的致畸效应；基因敲除动物资料证实，维生素A信号途径中的某些基因在二噁英诱发腭裂中的作用不同；体外实验显示，胚胎特异性因子Oct4缺失可阻断维生素A对二噁英信号途径的影响。我们为此推测：在腭裂诱发中，二噁英和维生素A信号途径间存在相互作用，Oct4可能是二者间的媒介分子之一。为阐释这一假设，我们拟选用各种体内外腭裂研究模型和多株基因敲除小鼠，借助免疫双标记、原位杂交、lacZ示踪、ChIP-qPCR和RNAi等技术，以次生腭发育期为切入点，探讨Oct4在二噁英和维生素A信号途径间的分子生物学作用模式，及其在腭突融合过程中对细胞迁移、分泌、分化和中嵴缝降解等的调节作用。研究资料可为围产保健提供指导。

选用各种体内外腭裂研究模型和多株基因敲除小鼠，借助免疫双标记、原位杂交、lacZ示踪、ChIP-qPCR和RNAi等技术，以次生腭发育期为切入点，探讨Oct4在二噁英和维生素A信号途径间的分子生物学作用模式。重要发现包括：对照组胎鼠在GD13-GD15腭突可正常上抬至水平方向，双侧腭突能够完全融合。TCDD组的胎鼠腭突发育瘦小，以致于两侧腭突无法接触融合。进一步研究结果显示，TCDD促进细胞的增殖以及细胞从G1向S期的发展进程，此外TCDD也增加了进入G2/M期细胞的数量。我们发现TCDD对人胎儿腭上皮细胞的增殖无明显影响。蛋白水平实验显示TCDD使cdk4、cyclin D1、cyclin E以及p21（Waf1/Cip1）表达增强但cdk2、bcl-2、cyclinB1和cyclinA的表达无明显变化。此外，TCDD激活了PI3K/AKT信号通路，PI3K拮抗因子LY294002部分抑制了TCDD诱导的细胞增殖和基因调控效应。TCDD暴露使CYP1A1 mRNA和蛋白水平增强，提示AhR信号的激活。用siRNA沉默AhR基因可抑制TCDD诱导的细胞增殖和PI3K/AKT信号通路的激活。其次，免疫组织化学染色、BrdU和TUNEL法结果显示：与对照组相比，TCDD可以抑制胎鼠腭突间充质的增殖相关蛋白表达，促进凋亡相关蛋白表达（P <0.05），同时在细胞实验中，TCDD可抑制腭突间充质细胞的增殖和活力，促进间充质细胞的凋亡（P <0.05）。Western Blot显示，TCDD可以抑制胎鼠腭突间充质中Oct4的表达蛋白的表达（P <0.05），亚硫酸氢盐测序法结果显示，TCDD在胎鼠腭突发育的关键时期并不改变Oct4启动子区甲基化水平（P >0.05）。TCDD可抑制RA信号途径相关分子的表达，蛋白免疫沉淀结果显示，TCDD可以促进Oct4与RARB、CRABPⅡ蛋白的结合。Oct4 siRNA 转染腭间充质细胞实验结果显示，Oct4被沉默后，TCDD对CRABPⅡ的抑制作用消失，随着TCDD浓度增加，CRABPⅡ表达表现为上调。总之，我们的研究资料证实，Oct4在TCDD诱发腭裂以及影响RA信号途径过程中起到关键作用。