外泌体参与上皮-间充质相互作用及其在牙再生领域的应用

The utmost goal of tooth regeneration is to generate a tooth organ with occlusal crown and regular root, as well as the whole functional structures. The genesis and regeneration of tooth requires an indispensable crosstalk between epithelium and mesenchyme and the transition of inductive signals. The traditional interaction pathways have been well studied including cell-cell contact, cell-extracellular matrix interaction and signaling pathway. Our preliminary data demonstrated that postnatal dental epithelium and mesenchyme stem/progenitor cells, when stimulated by control-released growth factors, such as BMP4/7 and Wnt3a, regenerated enamel-like and dentin-like tissues in two integrated layers. While there were no dental pulp formed and no structures such as crown and root observed. Exosome, as the signal mediator, can target to cells and induce sequential reactions. However, it is unknown that whether exosomes mediate the interaction between epithelium and mesenchyme. The overall goal of this project is to discuss a novel epithelium-mesenchyme crosstalk pathway which may be mediated through exosomes during tooth development and regeneration. The project objectives are to characterize exosomes in dental epithelial and mesenchymal, test the key factors mediating development process, take advantage of this model to induce postnatal stem cells differentiation and regenerate the whole tooth ultimately. Our research may contribute to discovering a novel mechanism of epithelium-mesenchyme interaction and optimize strategies for tooth regeneration by delivering the package of inductive signals.

全牙再生是形成兼具软硬组织结构及良好冠根形态的过程，组织再生效果取决于上皮-间充质二者间相互作用及诱导信号的传递。传统的上皮-间充质相互作用包括细胞直接接触、细胞基质作用及信号分子传导。本课题组前期研究发现，利用传统信号分子BMP4/7、Wnt3a可诱导成体牙源性干细胞再生形成牙釉质及牙本质层样结构，但缺少牙髓组织且无冠根形态形成。外泌体，作为一种新型信息传递载体，可特异性结合并作用于靶细胞，然而其是否参与上皮-间充质相互作用尚未知晓。本项目拟在前期研究基础上，探索上皮-间充质的新型相互作用方式，并将外泌体这种信号传递模式应用于牙再生。基于上述目的，本研究将首先验证外泌体是否参与牙齿发育过程；检测其功能及向上皮、间充质传递的关键信号；并进一步利用牙发育期特定的外泌体促进细胞定向分化，诱导成体干细胞实现全牙再生。本研究结果，有望补充对上皮-间充质相互作用机制的认识，并为牙再生提供一种新途径。

本项目基于牙齿发育的生理过程，围绕外泌体这种纳米级细胞外分泌囊泡，探索其作为一种独立的、可转移的遗传信号传递模式在器官发育中的作用；并进一步探索这种含遗传信号的囊泡在牙齿再生中的应用前景。本研究揭示了上皮—间充质相互作用的新机制，发现纳米级别细胞外囊泡（外泌体）是上皮—间充质细胞（牙齿发育过程中的主要两种细胞）间信号传递的重要介质，其可通过携带非编码RNA等生物遗传物质靶向调节WNT通路传递细胞间调控信号，进而参与发育过程；并在再生过程中诱导成体间充质细胞形成牙本质样结构。.本研究重要结果包括：1)首次在牙发育模型中鉴定外泌体的存在及性质；2)证明不同类型的靶细胞依赖不同的内吞机制可选择性的接受外泌体传递的信号；3) 分别从细胞、器官及转基因动物不同层面分析，证明外泌体在牙发育过程中的生物学功能；4) 分析上皮、间充质组织中外泌体不同的内容物，如蛋白及非编码RNA，并重点研究了外泌体中miRNAs的成分和功能；5）利用外泌体诱导人体牙髓间充质细胞再生形成牙本质。