蛋白聚糖通过调控FGFR2B通路启动小鼠牙再生的机制研究

The prospects for tooth regeneration in recent years are compelling. However, little was known about the role of Proteoglycan(PGs) in this regulatory network governing tooth development. In our previous study, we generated the PGs deficient mice and found that disrupting PGs in the dental epithelium of mice, revived tooth replacement capacity in monophyodont mice, accompanied with significant upregulation of FGF and ectopic expression of SOX2, a putative epithelial stem cell marker. This pilot study indicated that the dental epithelial PGs might regulate tooth replacement by mediating the homeostasis of dental epithelial Sox2 + stem/progenitor cells. To test this hypothesis, we will perform rescue experiments by inhibiting FGF signaling in the dental epithelium of PGs-deficient mice using a tet-on transgenic system. We will determine the mechanism of PGs regulation on FGF pathway through in vitro experiments using Fgf2b-engineered cell line. Moreover, we will build the transgenic mice model of cell lineage tracing, cell ablation and tissue recombination and transplantation to determine the role and molecular mechanism of the dental epithelial PGs mediated the homeostasis of dental epithelial Sox2 + stem/progenitor cells in the replacement tooth formation. This study will reveal the mediating mechanism of PGs during tooth development and regeneration, and also provide novel insights for promoting the clinical application of biological tooth regeneration.

牙再生成为国内外组织工程学领域近年来的研究热点，但针对细胞外蛋白聚糖（Proteoglycans，PGs）在该过程中的分子调控机制研究甚少。申请人在前期建立的牙上皮PGs缺陷小鼠模型中，意外发现牙上皮PGs功能障碍可致小鼠萌出多生的继承切牙，并伴有FGF通路及牙胚上皮干细胞标记物SOX2的表达上调。提示PGs可能通过调控FGF通路影响牙上皮Sox2+谱系干细胞稳态，从而启动牙再生。本项目拟利用四环素诱导转基因小鼠模型，通过抑制PGs缺陷鼠牙上皮内FGFR2B受体活性尝试逆转多生牙表型；并结合稳定转染Fgfr2b的细胞系共同揭示PGs与FGF通路间的作用机制；随后利用细胞谱系示踪、细胞消融以及体外牙胚重组技术共同探究Sox2+谱系干细胞在PGs缺陷小鼠继承牙形成中的作用机制。本研究将明确PGs在牙齿再生中的调控机制，为阐明牙齿发育的分子机制提供新的见解，为临床牙再生的应用提供新的思路。

蛋白聚糖（PGs）是一类特殊的糖蛋白，作为重要的细胞外信号分子，参与调控大部分组织器官的胚胎发育。申请人在前期建立的牙上皮PGs缺陷小鼠模型中，意外发现上皮PGs缺陷可致小鼠出现多生切牙表型，并伴有FGF通路及牙胚上皮干细胞标记物SOX2的表达上调。提示PGs可能通过调控FGF通路影响牙上皮Sox2+谱系干细胞稳态，从而启动牙再生。本项目的主要研究内容和结果包括：1、成功构建四环素诱导转基因小鼠模型，证实抑制PGs缺陷小鼠牙上皮内过表达的FGF通路活性能够逆转再生切牙表型，明确FGF10/FGFR2B是PGs缺陷小鼠再生牙发生的关键信号通路。2、结合体外构建的稳定转染Fgfr2b的细胞系揭示了PGs通过限制FGF10的梯度扩散，发挥对FGF10/FGFR2B通路的调控作用，且其作用与糖胺聚糖链的浓度与硫酸化程度密切相关。3、利用细胞谱系示踪、细胞消融以及体外牙胚重组技术，发现Sox2+谱系干细胞可能是PGs缺陷小鼠继承牙发生的细胞来源之一，且Sox2+谱系干细胞通过旁分泌途径参与多生牙的形成。本项目系统地阐明了PGs通过调控FGF10/FGFR2B通路，调控Sox2+谱系干细胞参与牙胚的再生发育的分子机制，明确了PG在牙齿发育再生中的调控作用，为完善牙发育的分子信号网提供新的依据。本项目发表SCI论文1篇，发表北大中文核心期刊论文2篇。项目负责人应邀在国内外会议上进行壁报展示及口头报告，获得最佳设计奖。在3年的项目执行期内，本项目严格按照经费预算，顺利完成了预期目标。