胞外基质蛋白Hemicentin1在牙本质形成中的调控作用

Tooth development depends on reciprocal epithelial-mesenchymal interactions (EMI). Studying the molecular mechanism of this process has an important significance in tooth repair and regeneration. The existing data inform that different sets of genes and proteins show distinct temporal and spatial expression patterns during tooth development. In our previous study, both Hmcn1 gene and Hemicentin1 protein were found to be differentially expressed on the crown vs. root side in epithelial and mesenchymal tissues. In vitro studies demonstrated that Hmcn1 was down-regulated during the proliferation and differentiation of odontoblasts. The data inform the following hypothesis: Hemicentin1 negatively regulate the differentiation of odontoblasts during dentinogenesis. This hypothesis will be tested by the following strategies: Recombinant Lentivirus vector encoding Hmcn1 cDNA will be constructed to infect human dental pulp cells (hDPCs). Proliferation and differentiation assay will be performed on hDPCs with Hmcn1 overexpressed and knocked down. PCR array will be utilized to identify Hmcn1 associated genes and signal pathways. Results will be verified in vitro. The potential capability to induce differentiation of oral epithelium will be further examined. The study of regulatory mechanism of extracellular matrix Hemicentin 1 during dentinogenesis will provide a new level of understanding of EMI signal network, and will provide new strategies for regeneration therapies and engineering applications of teeth.

牙发育依赖于上皮-间充质的相互作用，研究阐明其分子机制对牙体组织缺损的修复和再生具有重要意义。一些在牙发育过程中具有重要作用的基因和蛋白质在牙发育的不同阶段呈现出不同的时空表达特点。课题组前期研究首次发现，胞外基质蛋白Hemicentin1（Hmcn1）在小鼠冠、根部牙本质中的表达水平有显著差异，体外实验证实Hmcn1在牙髓细胞的增殖和分化过程中表达受到抑制，可能通过负向调节参与牙本质的形成。本课题拟利用慢病毒载体在人牙髓细胞中过表达Hmcn1或敲低Hmcn1的表达水平，检测对人牙髓细胞增殖和分化的影响，通过PCR array筛选Hmcn1调节相关的基因并进行验证，并进一步研究其在体外和体内环境中对口腔上皮的分化诱导的能力，从而研究Hmcn1在牙本质形成中的调控作用并探索其分子机制，完善牙本质形成过程中的上皮-间充质相互作用的信号调控网络，为牙的组织工程修复再生提供研究基础。

本研究在课题组前期研究的基础上，针对在小鼠切牙和磨牙冠部及根部差异表达的胞外基质蛋白Hemicentin 1（HMCN1）进行了初步研究，在体外用基因重组技术构建重组慢病毒，利用慢病毒载体介导基因转移方法在人牙髓细胞中敲低Hmcn1的表达水平，检测其改变后对人牙髓细胞增殖和分化的影响。