牙胚发育中Mint调控上皮-间充质相互作用机理的研究

During the tooth development, the molecular mechanism of epithelial-mesenchymal interactions is unknown. It is a serious bottleneck to restrict the tooth regeneration. The key gene function is unknown, which obstructs the research progress of molecular mechanisms during tooth development. Mint is a newly discovered transcription factor. Mint has been reported to act as a suppressor of Notch signaling, which plays an important role in tooth development. Mint enhances Cbfα1 activation of multiprotein complexes assembled by the OCFRE. Msx2 targets this complex as a mechanism of transcriptional inhibition. In osteoblasts, Mint may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses. Previous data have shown that enamal knot and dental papilla fail to form in Mint-/- tooth germ. Although these results indicate that it is a key modulating gene, it is still unknown that the mechanism how Mint regulates reciprocal epithelial-mesenchymal interactions during the tooth development. In order to gain an insight into the molecular mechanism, We use Mint knockout mice embryos for organ culture and tissue culture to investage the changes in molecular expression and tooth germ. Then gene expression analysis using in situ hybridization will be combined with classical tissue recombination and bead implantation experiments to examine the functions of Mint in sequential and reciprocal epithelial-mesenchymal interactions. So we will ascertain that Mint is a key gene, modulating sequential and reciprocal epithelial-mesenchymal interactions during tooth development. All these results will give more groundwork for an insight into the molecular network in tooth development and will provide fundamental knowledge for the realization of tooth regeneration in the near future.

牙胚发育中上皮-间充质相互作用的分子调控机理不明是严重制约牙再生的瓶颈之一。关键基因功能不明制约了牙胚分子调控机理的研究进展。Mint是新近发现的转录调控因子，是牙胚发育关键信号Notch负调控蛋白，可与牙胚关键转录因子Msx2、Cbfα1作用，调节矿化相关蛋白骨钙素的转录。前期研究发现：Mint基因敲除小鼠牙胚釉结、牙乳头不能正常形成，提示该基因是调控牙胚发育关键基因，但它在牙胚中的功能和对上皮-间充质相互作用的调控机理并不清楚。本项目利用Mint基因敲除小鼠，结合器官培养、组织培养技术研究Mint敲除后牙胚畸形变化和相关信号分子表达，确定其参与的信号通路；采用组织重组技术研究Mint调控成牙潜能在上皮和间充质间转移的分子机理；从而明确Mint是牙胚发育中调控上皮-间充质相互作用的关键调控因子，为牙齿发育分子网络调控体系提供新的理论基础，为牙再生中诱导牙齿发育重新启动的策略提供实验依据。

牙再生是近年研究的热点课题，但牙胚发育中上皮-间充质调控机理不明是制约牙再生的瓶颈之一。成牙关键基因和蛋白质功能不明制约了牙齿发育调控机理的研究进程。探明未知基因和蛋白质的功能，明确参与调控的信号通路，是揭示牙胚发育分子调控机理的关键组成。Mint就是新近发现调控牙胚发育的新基因。研究发现Mint基因敲除小鼠牙胚釉结、牙乳头不能正常形成，提示该基因是牙胚发育关键基因，但它在牙胚的功能和对上皮-间充质调控机理并不清楚。.本课题利用Mint基因敲除和条件性敲除小鼠，结合器官培养、组织重组、细胞培养等研究Mint在牙胚发育中的分子机理。.第一部分： ① Mint基因敲除牙胚发育异常，无牙胚结构，组织排列紊乱。② 釉结标志分子p21、shh mRNA表达几乎阴性。.第二部分：① 组织取自E13前，在上皮、间充质重组体中，上皮基因型Mint+/+，均有牙胚形成；上皮基因型为Mint-/-重组体中未见牙胚形成。② 组织取自E13后，无论是Mint-/-上皮与Mint+/+间充质重组体，还是Mint+/+上皮与Mint-/-间充质重组体都可以观察到牙胚形成。③ 分离E12.5 Mint-/-牙胚上皮和间充质，将Mint-/-间充质与Mint+/+上皮重组，36-48h，Mint+/+上皮换成Mint-/-上皮，移植到裸鼠肾被膜下培养2 w，重组体中依然有牙胚形成。.第三部分：① Mint在上皮和间充质内源性表达，在单独培养的上皮Mint表达减弱。② Msx2和Bmp4可以诱导Mint表达，提示Mint是Msx2、Bmp4下游信号分子。.第四部分：① Mint条件性剔除牙胚发育晚期，成牙本质细胞排列紊乱，牙本质层较薄，未见矿化基质。② 体外培养牙乳头细胞Wnt1-Cre/Mint-/-细胞活性明显减弱，ALP、OC较低。③ pEF-BOS-Mint质粒转染牙乳头细胞，细胞活性增强，ALP、OC增加。④ Mint条件剔除后，矿化相关蛋白DSPP、DMP1、DSP表达减弱。.本课题证实Mint是牙胚发育关键基因，牙胚正常发育依赖Mint在上皮诱导成牙能力，明确Mint诱导成牙能力主要存在于上皮中，并有时效性的。牙胚发育信号一旦启动，即当Mint成牙能力从上皮转移到间充质，Mint在上皮的功能对牙胚生长发育就不是必需的，但仍影响成牙本质细胞、成釉细胞增殖分化，以及牙本质的形成、矿化。