Notch通路相关基因甲基化对牙发育的调控机制研究

Tooth loss is commonly observed in clinic and can intensively affect a patient` life quality. Current restorative or prosthetic therapies cannot fulfill the clinical needs due to various limitations. On the other hand, regenerated tooth fabricated by tissue engineering approaches may provide a novel and practical solution. To achieve this, it is essential to understand the regulation mechanism of tooth development. It has been shown that Notch signaling pathway plays an important role in tooth development. In our previous studies, we found that the methylation statuses of Notch-associated genes are related to tooth agenesis. Based on these results, we hypothesize that during embryonic development, high methylation of Notch-related genes may influence the phenotype of odontogenic epithelial cells (EC), thus hinder the proliferation and differentiation of ECs as well as their interaction with mesenchymal cells. To test this hypothesis, our group intends to design a targeted methylation system through sgRNA-Cas9-Dnmt3a-3l expressing recombinant plasmid. We intend to methylate multiple targets of Notch signaling in odontogenic epithelial cells, and measure tooth development potential in target cells in multiple aspects, including the cell phenotype, cell interaction and tissue organ development. This proposal will investigate the role of Notch pathway in regulating odontogenic ECs in tooth development on the level of DNA methylation, and aims to probe into the regulation mechanism. The potential results will provide experimental evidences for tooth regeneration and epigenetic researches.

牙缺失是人类的常见病、多发病，严重影响患者的生活质量。利用组织工程技术实现有生物活性的牙再生可以改善现有修复方法的不足，其中明确牙发育调控机制是关键。以往的研究表明以Notch为中心的信号通路的相关基因对牙发育起着重要的调控，然而这些基因甲基化对牙发育的调控未见研究报道。本课题组前期研究发现Notch信号通路相关基因的甲基化状态可能与牙齿发育异常密切相关，我们推测胚胎发育中的Notch通路相关基因的高甲基化状态会影响牙源性上皮细胞的表型，进而损害其发育成牙胚的能力。因此本课题组拟采用sgRNA-Cas9-Dnmt3a-3l重组蛋白表达质粒对牙源性上皮细胞的多个Notch通路相关分子进行靶向甲基化，从细胞表型、细胞相互作用、组织器官发育等多个层面观察靶细胞成牙能力的变化。从DNA甲基化的层面上探索Notch通路对牙源性上皮细胞的影响及调控作用，力求为牙发育的表观遗传学研究提供科学的实验依据。

牙缺失是人类的常见病、多发病，严重影响患者的生活质量。以往的研究表明以Notch为中心的信号通路的相关基因对牙发育起着重要的调控，本课题组前期研究也发现Notch信号通路相关基因的甲基化状态可能与牙齿发育异常密切相关，我们推测胚胎发育中的Notch通路相关基因的高甲基化状态会影响牙源性上皮细胞的表型，进而损害其发育成牙胚的能力。为了研究DNA甲基化对特定基因表达的影响，我们拟采用sgRNA-Cas9-Dnmt3a-3l重组蛋白表达质粒对牙源性上皮细胞的多个Notch通路相关分子进行靶向甲基化，从细胞表型、细胞相互作用、组织器官发育等多个层面观察靶细胞成牙能力的变化。在本研究周期内，我们构建了含特定启动子区段的，表达红色荧光的靶细胞，先后尝试了使用质粒、慢病毒、腺病毒为载体的转染体系。研究结果表明质粒共转染法和慢病毒+腺病毒转染法细胞毒性过大。应使用先慢病毒转染dCas9-dnmt3ac片段，筛选出阳性克隆后再转染sgRNA的方法来进行基因的甲基化。目前的系统中，基因靶向甲基化对目的基因的抑制效果很低，与临床观察的结果不符，其原因可能与哺乳动物体内，甲基化的DNA区域较易被MeCP2这样的DNA结合蛋白所识别，并募集组蛋白脱乙酰化酶和组蛋白甲基化酶有关。后继的研究将1.针对以往的体外研究样本做进一步的验证，确认DNA甲基化区域是否同时存在组蛋白脱乙酰化和组蛋白甲基化的现象。2.寻找体外研究中模拟组蛋白脱乙酰化和组蛋白甲基化的方法。