Cdc42通过Rheb/mTOR介导自噬调控牙源性上皮细胞凋亡的机制研究

Cell apoptosis plays a pivotal role in the development of dental epithelium. Our previous work showed that epithelial Cdc42 deletion induced increased autophagy and apoptosis within dental epithelial cells, leading to tooth development defects in mice. PCR array for autophagy marked the high expression level of genes which are co-regulators of autophagy and apoptosis. Moreover, mTOR pathway which regulates autophagy as known and its upstream regulator Rheb were attenuated by Cdc42 deletion. Further in vitro studies demonstrated knockdown of Cdc42 in the dental epithelial cell line caused increased autophagy and cell apoptosis, but inhibition of autophagy rescued cell apoptosis induced by Cdc42 knockdown. Pieces of evidence above pointed out our hypothesis Cdc42 may regulate cell apoptosis by mediating autophagy in dental epithelium through Rheb/mTOR pathway. In this project, we generated Cdc42 conditional knockout mice models. By using these mice models, firstly, we will investigate the spatiotemporal role of mTOR in regulating apoptosis by mediating autophagy in dental epithelium by double immunofluorescence staining. And then the mechanism of which how Cdc42 interacted with mTOR signaling will be determined by proteomics analysis. Finally, in-vivo and in-vitro interruption of tooth germs will be carried out to demonstrate how Cdc42-mTOR signals regulated cell apoptosis by mediating autophagy in dental epithelium during tooth development. As we start with a new perspective that autophagy could regulate cell apoptosis in the dental epithelium, this project will demonstrate the role of Cdc42-mTOR signals in tooth development and provide a novel insight into the mechanism of tooth development.

细胞凋亡是牙源性上皮发育的重要生理过程。前期研究发现敲除Cdc42引起牙源性上皮细胞自噬与凋亡增加，导致小鼠牙发育缺陷。自噬PCR芯片发现敲除Cdc42可上调细胞中自噬-凋亡的调控基因，并抑制相关通路mTOR活性和其上游调控蛋白Rheb的表达；进一步体外实验证实敲低Cdc42引起牙源性上皮细胞系自噬-凋亡增加，抑制自噬则凋亡减少。提示Cdc42可能通过Rheb/mTOR介导自噬调控牙源性上皮细胞凋亡。本项目将在Cdc42条件敲除鼠的模型中，首先通过免疫荧光双标共定位的方法明确mTOR介导自噬在凋亡细胞中的时空作用，并结合蛋白质组学等分析Cdc42与mTOR的结合方式；在此基础上通过体内体外牙胚干预等实验阐明牙发育中Cdc42-mTOR信号网络介导自噬调控凋亡的机制。本研究以自噬调控牙源性上皮凋亡为切入点，有望揭示Cdc42-mTOR信号网络在牙发育中的作用机制，为明确牙发育机制提供新思路。

牙源性上皮是成牙信号的始发组织，是牙发育及牙再生研究的关键组成部分。牙源性上皮细胞发育异常可导致不同类型的牙发育异常/缺陷。牙源性上皮在出生前主要形成成釉细胞，分泌牙釉质覆盖在牙本质表面。在出生后，牙源性上皮逐渐形成上皮根鞘，引导牙乳头、牙囊组织等间充质组织逐步形成牙根。研究表明，出生后的牙源性上皮缺乏重要的因子如Wnt10a等，会造成“牛牙症”的发生。可见，牙源性上皮不止在胚胎期发育，在出生后仍发挥调节牙发育和牙萌出的作用。研究牙源性上皮细胞在胚胎期和出生后的调控机制，可认识牙发育进程、了解牙发育异常机制，还可为牙再生策略供思路。前期研究发现，牙源性上皮（成釉器）特异敲除Cdc42或mTOR引起成釉器囊性变、牙胚发育异常、成釉细胞分化异常，但其机制尚不清楚。本项目围绕Cdc42/mTOR信号网络如何调控牙源性上皮干细胞的稳态、分化及再生开展了一系列研究。首先构建药物诱导上皮特异敲除Cdc42或mTOR的小鼠，并通过microCT等分析敲除鼠的切牙、磨牙的表型；在成年鼠中，使用他莫昔芬诱导敲除Cdc42或mTOR，而后构建切牙切断再生模型，分析和对比Cdc42敲除鼠和mTOR敲除鼠的釉质再生能力、牙根再生能力；通过蛋白质组学分析他莫昔芬诱导敲除Cdc42或mTOR的牙源性干细胞的功能并进一步验证；最后，分离Cdc42或mTOR敲除鼠的切牙颈环处牙源性干细胞，分析其ROS状态，明确Cdc42/mTOR调控牙源性上皮干细胞发育及再生的机制。同时，为明确出生后牙根发育的机制，本项目进一步通过RNA芯片等技术去探讨牙囊干细胞的发育及再生机制。目前相关研究结果已发表SCI论文5篇，会议论文1篇，参加国际会议进行学术交流1次，培养3名硕士生毕业和3名在读研究生。