p75+颌突间充质干细胞的体外分化命运与成牙分化潜能的初探

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, playing key roles in craniofacial development. CNCCs are highly migratory multipotent cells that give rise to diverse derivatives, such as peripheral nerve cells, skin melanocytes, and cranial mesenchymal cells.Following proper epithelial-mesenchymal interactions, postmigratory CNCCs differentiate into multiple phenotypes and contribute to the formation of various head and neck structures, especially tooth development. However, details about their in vitro properties and functions are still incomplete, and the characterization regarding their reaction to dental epithelium is not fully understood. Their in vitro harvest, fates and precise roles in tooth initiation remains controversial. In this project, we have harvested ectomesenchymal stem cells (EMSCs) from E11.5 SD rat embryonic facial processes by the traditional enzymatic dissociation method and then purify them by fluorescence activated cell sorting (FACS) to obtain p75 neurotrophin receptor (p75NTR) positive EMSCs (p75+ EMSCs) in our primary study. Their homogeneity, in vitro stability, and multiple lineage differentiation ability were investigated. The results indicated that p75+ EMSCs showed promise as an alternative stem cell resource, with a potential application to tissue engineering, especially craniofacial and dental tissue engineering. Base on this primary study, the cranial neural crest origin of p75+ EMSCs will be further identified in the following experiments, in order to establish an in vitro cell model for CNCCs, by which we can discuss the in vitro fates, influence factors, potential odontogenesis of CNCCs. In addition, the changes in the expressions of odontogenic genes, such as Hes1、Mash1、Dlx1/2、Dlx5/6、Barx-1、Msx-1 and so on , are investigated when p75+ EMSCs interact with dental epithelial cells in vitro, to determine the precise roles of CNCCs in epithelial-mesenchymal interaction which triggers tooth initiation. This project will provide not only the in vitro data for revealing the precise mechanism under tooth development but a promising stem cell source for tooth regeneration.

颅神经嵴细胞属多能干细胞，具有向多种组织细胞分化的潜能，在胚胎发育早期从神经管迁移并定居于上下颌突，是颅颌面、特别是牙齿发育的重要细胞来源。目前对于它的生物学特性认识还远远不够，其体外获取方法、分化命运以及成牙启动机制等问题还存在争议。本课题采用酶消化贴壁法从SD大鼠胚胎的颌突组织中分离培养间充质细胞，再通过流式分选获得单一的p75NTR 阳性外胚间充质干细胞（p75+EMSCs），明确p75+EMSCs的颅神经嵴来源，建立颅神经嵴源性外胚间充质干细胞的体外研究模型；并在此基础上进一步探讨p75+EMSCs的体外分化命运与影响因素、成牙分化潜能以及在牙源性上皮细胞信号因子作用下成牙关键基因（如Hes1、Mash1、Dlx1/2、Dlx5/6、Barx-1、Msx-1等）的表达变化，揭示牙发育启动时外胚间充质- - 上皮相互作用的确切机制，同时为细胞重聚技术获得牙齿再生提供新的、理想的种子细胞。

颅神经嵴细胞属于多能干细胞，具有向多种组织细胞分化的潜能，在胚胎发育早期从神经管迁移并定居于上下颌突，是颅面部特别是牙齿发育的重要细胞来源。目前对于其生物学特性的认识不够，其体外获取方法、分化命运以及成牙启动机制等问题还存在争议。因此研究牙发育启动时上皮-间充质的相互作用确切机制，为细胞重聚技术获得牙齿再生提供新的、理想的种子细胞有重要意义。.本课题以牙源性干细胞的共同始祖-颅神经嵴源性的颌突外胚间充质干细胞作为主要研究对象，利用流式细胞术对原代细胞进行分选，获得形态均一，在体外培养具有稳定增殖能力，且在多项诱导下（成脂，成骨，成软骨，成神经）能够顺利分化的干细胞特性的p75阳性外胚间充质干细胞。在成肌及牙源性非胶原酶/牙囊细胞条件培养基诱导下，通过检测myogenin, myosin发现其能够分化为成肌细胞，并且在细胞膜片中能够检测到MyoD基因，动物实验中能够修复肌肉损伤；通过检测BSP，Col1，ON，OCN，DSPP及CAP等基因，发现其能够成功分化为成牙骨质细胞。通过对p75阳性和p75阴性外胚间充质干细胞成牙本质分化能力的对比以及DSP，DMP1，ALP基因的检测，发现p75阳性外胚间充质干细胞成牙本质分化能力更强，并且在异位植入免疫缺陷型小鼠体内的细胞膜片中也证明了体外实验的观点，p75阳性和p75阴性外胚间充质干细胞基因芯片的检测及抑制剂si-Smad4和激动剂Kartogenin对细胞的处理，表明Smad4能够正向调控细胞的成牙本质能力。.通过系列研究，本课题组不仅建立了稳定且可靠的研究牙发育上皮-间充质相互作用的颅神经嵴源性细胞模型---p75+EMSCs，并且成功探讨了其体外分化包括：成肌、成牙骨质、成牙本质的多项分化潜能。进一步探索了影响其成牙本质分化能力可能存在的分子机制。初步揭示了牙齿发生、发育的潜在机制。为牙齿发育和组织工程牙的研究提供了良好的理论依据以及实验基础。