TCDD慢性暴露与母体免疫功能在腭裂发生中的交互作用及其分子机制研究

The pathogenesis of cleft lip and palate is relatively complex, in addition to environmental and other factors, some scholars found that the experimental animal model for immune stimulation can obviously reduce the occurrence of cleft palate, therefore, the occurrence of cleft lip and palate is also associated with the maternal immune function. For the interaction mechanism between maternal immune function and environmental factors in the formation of cleft palate is unclear, a lack of chronic exposure animal model simulating the actual incidence of environmental factors presently.Dioxin (TCDD) in many dubious environmental factors in the formation of cleft lip and palate is the most clear, the strongest teratogenicity.So we plan to apply Laser Micro-Dissection and Real-time RT-PCR and other technology to study the spatiotemporal expression pattern of the key gene signal pathway in the fusion process of embryonic palate in the two statement of “chronic TCDD exposure ”and “chronic TCDD exposure+ immunologic stimulant” effcting C57BL/6J mice based on the former established chroni TCDD exposure-induced cleft palate mice in this project ,and we would also observe the influence of immunologic stimulant（GM-CSF, IFNγ）on maternal immunity and the development of embryonic palate andresearch its interventional mechanism at the same time. By this way we want to illuminate how the interaction between chronic TCDD exposure and maternal immunity would influence the fusion of embryonic palate, and find a new way of preventive intervention on high risk groups of cleft palate under unfavourable environment.

唇腭裂的发病机制较为复杂，除环境等因素外，有学者发现对于实验动物模型给予免疫刺激可明显降低腭裂的发生，因此唇腭裂的发生还与母体的免疫作用相关。目前对于母体免疫功能与环境因素在腭裂发生中的交互作用机制尚不明确，更缺乏模拟实际发病过程的环境因素慢性暴露的动物模型。二恶英（TCDD）在众多可疑影响唇腭裂发生的环境因素中最为明确、致畸性最强。本项目通过前期建立的TCDD慢性暴露诱导的小鼠腭裂模型，拟在TCDD慢性暴露和TCDD慢性暴露+免疫刺激剂作用于C57BL/6J小鼠的两种环境情况下，应用激光显微切割技术、荧光定量PCR等技术，按时空顺序研究腭胚突融合阶段关键基因信号通路的变化规律，并观察免疫刺激剂（GM-CSF, IFNγ）对母体免疫功能和腭胚突发育的影响与干预机制。阐明二恶英慢性暴露与母体免疫功能交互作用对腭胚突融合影响的机制，探索不利环境情况下对腭裂高危人群干预性预防的新途径。

唇腭裂的发病机制较为复杂，除环境因素外，还涉及遗传以及母体的免疫功能。二恶英（TCDD）在众多可疑影响唇腭裂发生的环境因素中最为明确、致畸性最强。目前关于腭裂模型的建立，常常用单一药物致畸，模型常不稳定，本项目在前期的研究基础上，利用高剂量的二噁英与地塞米松（DEX）联合一次性诱导，成功构建了稳定的腭裂模型，孕鼠流产率及死胎率均达到较低水平。在实验第二部分运用Real-time PCR及Western-blot等分子生物学技术发现，在腭融合关键时期，TCDD+DEX诱导TGF-β3表达下降，Alk5表达升高，差异有统计学意义（P＜0.05），提示其与腭裂的发生具有一定的相关性。本项目第三部分在体内采用VitB12与免疫刺激剂（GM-CSF）联合作用，试图逆转TCDD与DEX诱导的腭裂模型，实验结果未能获得成功，然而对TCDD+DEX诱导的中嵴上皮细胞的细胞形态改变有一定抑制作用，并对TCDD+DEX诱导的信号表达改变也有抑制作用，且提高了母体的免疫功能。本项目第四部分，在体外实验中进行腭胚突培养，利用慢病毒转染，导入外源性TGF-β3，成功逆转了TCDD+DEX诱导的腭裂模型，为腭裂的治疗提供了新的思路。