p75NTR在颅神经嵴源性颌突外胚间充质干细胞成牙分化中的调控作用与机制研究

Fully functional bioengineered tooth is no doubt the most optimal method to replace the missing teeth in future. However, the molecular biological mechanism under tooth morphogenesis and development is still not clear, which greatly limits the progress of tooth regeneration. Cranial neural crest-derived cells (CNCCs), a precursor cell population arising from the neural folds of the developing embryo, migrate to and populate the branchial arches, giving rise to many tissues and organs in the craniofacial region, including bones, teeth, and muscles. The postmigratory CNCCs, following the interaction with dental epithelium, differentiate into various pre-odontoblasts, such as dental papilla cells and the dental follicle, subsequently forming dentine, pulp, cementum, and periodontal ligaments. Therefore, they are considered as a superior one in the studies of dental stem cells to reveal the mechanism under tooth morphogenesis. But the morphologic heterogeneity and tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro without leukemia inhibitory factor (LIF) limit the further research on the characterization of postmigratory CNCCs. In order to solve this problem, p75 neurotrophin receptor (p75NTR), a typical neural crest marker, were used in our previous studies to purify the postmigratory CNCCs as p75NTR positive ectomesenchymal stem cells (p75+EMSCs) by fluorescence activated cell sorting. p75+EMSCs, which were tested to be homogeneous in shape and stable in vitro without any LIF, is a good stem cell model for the studies on tooth development. Basing on the literature review and our previous research, especially the finding of the strong expression of p75NTR in the cells locating in dentino-enamel junction at the bell stage of tooth development, we firstly investigate the expression rules of p75NTR and its related regulatory factor in p75+EMSCs, revealing the roles of p75NTR in the proliferation, mineralization activity and odontogenesis of p75+EMSCs in the present project. The aim of this project is to contribute to revealing molecular biological mechanism under tooth development and to promote the fully functional bioengineered tooth regeneration.

组织工程化牙齿无疑是未来解决牙齿缺失最为理想的手段。然而，牙齿发生、发育过程中的确切分子生物学机制仍不完全清楚是目前制约组织工程化牙齿发展的瓶颈。颅神经嵴源性外胚间充质干细胞是除牙釉质以外所有牙齿组织的生成细胞，被认为是研究牙发育机制理想的干细胞群；然而，细胞异质性和体外自然分化倾向限制了其在该领域的应用。为此，我们采用神经营养因子受体p75NTR对该干细胞群进行流式分选，获得了细胞形态均一、能代表高纯度神经嵴源性的p75NTR阳性外胚间充质干细胞（p75+EMSCs），提供了一个良好的体外细胞模型。本课题基于文献回顾、前期研究成果、以及预实验中p75NTR在牙发育钟状期釉牙本质界细胞中强表达这一发现，首次探讨p75NTR及其相关调控因子在p75+EMSCs中表达规律、以及在细胞增殖、矿化、成牙分化中的调控作用，为揭示牙齿发生、发育的分子生物学机制提供实验参考依据，推进组织工程化牙齿进程。

组织工程化牙齿是目前大家公认的解决牙齿缺失问题最为理想手段，但牙齿发育过程中的众多复杂的分子生物学机制尚不清楚，严重制约了组织工程化牙齿的进展。因此，揭示牙发育过程中的分子生物学机制具有重要的科学意义。.本项目通过流式检测细胞表面标志物获取并验证了能在体外代表颅神经嵴源性外胚间充质干细胞（EMSCs）的模型——p75+EMSCs，通过mRNA水平和蛋白水平，检测ALP、RunX2、Osterix，证实EMSCs具有良好的矿化能力。进一步使用siRNA沉默P75NTR或pLJM1质粒过表达P75NTR，发现P75NTR、矿化关键基因ALP、RunX2、Osterix，随着成骨诱导时间的延长，逐渐增高，说明P75NTR对EMSCs的矿化能力具有促进作用。首次发现Mage-D1可能以“变阻器”的角色参与p75NTR调控EMSCs的矿化过程，提出p75NTR—Mage-D1—Dlx/Msx信号机制假说，为后续研究提供了良好的科学问题。.通过腹部手术获取同一SD孕鼠胚胎发育不同时间点（12.5d/19.5d）的EMSCs，使用Affymetrix表达谱芯片检测12.5dEMSCs和19.5dEMSCs中差异基因表达，我们发现SOST在19.5dEMSCs中高表达（36倍），GOpathway分析wnt信号通路在其中起重要作用。进一步借助siRNA沉默、pLJM1-P75NTR及pMYC-SOST，率先发现P75NTR负向调节SOST，并通过抑制SOST的功能上调wnt通路活性，进而促进EMSCs的矿化能力。通过动物实验研究发现，P75NTR基因敲除小鼠股骨的骨量、骨小梁密度均少于野生型小鼠，并通过钙黄绿素检测牙齿周期性矿化情况，证实P75NTR对骨组织矿化的抑制作用，并初步说明P75NTR调控生物节律性和牙齿硬组织的周期性矿化。.本课题证实了P75NTR在EMSCs中对矿化能力的促进作用；探讨了P75NTR参与牙齿发育过程的分子生物学机制，为阐明牙齿形态发生、发育的分子生物学机制、推进组织工程化牙齿进展提供新的思路和实验参考依据；并初步探明p75NTR调控生物节律性和牙齿硬组织周期性矿化的证据，进一步揭示牙齿发生、发育机制。