初级纤毛介导的Shh信号与小鼠腭发育关系的研究

Shh signaling play a important role during palatal development,but the transduction of of Shh downstream signaling remained unknown.Studies showed transduction of Shh signaling dependent on primary cilia in some kinds of tissue in mammalian and primary lilia involved in regulating cell cycle and response to environment.Applicant discovered that expression of Shh signaling and proliferation of mesenchyme at mouse embryonic palate was changed after timed pregnant mice was treated with DEX and VitB12.Applicant make a hypothesis that transduction of Shh signaling in mesenchyme of mouse embryonic palate dependent on primary cilia. That Environment factors regulate transduction of Shh signaling by effection ciliary components, which control mesenchymal cells cycle. So the object of the study is to detect the pattern of Shh signaling transduction in mouse palatal embryonic cells and change of environment factors,such as DEX, make effect on Shh signaling transduction,cells cycle and dynamic change of cilia by immunohistochemistry, scanning electron microscope, LSCM, RNAi, and so on. To explore the mechanism of evironment factors influence palatal development from a new perspective.

Shh信号通路在腭发育中有重要作用，但其下游信号如何传递尚不清楚。研究证实哺乳动物多种组织中纤毛可传递Shh等信号因子，同时参与调控细胞周期，也可对外环境的改变发生相应变化。申请者前期研究发现地塞米松和维生素B12作用于特定孕期母鼠后，胎鼠腭板Shh信号表达和间充质细胞增殖均发生改变，基于这一发现，申请者假设小鼠腭胚突间充质细胞Shh信号传导依赖初级纤毛，外界环境因素可通过影响纤毛蛋白组件调控Shh下游信号因子传递，二者协同调节间充质细胞周期，从而影响腭的发育。故本课题利用细胞体外培养系统，运用免疫组织化学、扫描电镜、激光共聚焦显微镜和RNAi等技术检测Shh信号在小鼠腭胚突间充质细胞中的传递模式，观察外界因素干预下，如地塞米松等对Shh信号传递、细胞周期和纤毛动态变化的影响，探讨它们之间的关系，从一个新的视角研究环境因素影响腭发育的机制。

Shh相关信号通路在哺乳动物腭发育中有重要作用，但该信号通路在腭发育过程中是否依赖初级纤毛发挥作用尚不确定。本研究拟体外培养小鼠腭上皮及间充质细胞，通过免疫荧光等手段观察小鼠腭上皮或间充质细胞中是否存在初级纤毛，进一步沉默体外培养小鼠腭细胞初级纤毛关键组件，IFT88和IFT122，加入外源性Shh蛋白后通过免疫印迹和激光共聚焦观察Shh下游信号Ptc1、Smo的变化，并通过检测Gli3和Cyclin D1和观察Shh信号通路是否能激活其下游基因转录引发细胞增殖效应，检测初级纤毛解聚因子HEF1和Aurora A，探讨初级纤毛转运系统关键组件失活后，对初级纤毛的影响，同时探讨地塞米松干预后，Shh信号相关因子的表达变化和是否通过影响初级纤毛转运系统关键组件和其解聚来阻止Shh信号的传递，为哺乳动物腭发育过程中Shh信号作用的机制提供新思路。