转录因子Barx1调控Dlx5介导PDA促正畸牙骨质再生的分子机制

Developing an effective strategy to promote cementum regeneration has important clinical implications for adult orthodontic treatment. Applicants have previously found that polydopamine (PDA) can effectively promote cementum regeneration and enhance the differentiation of cementoblasts. Then the high-throughput methods such as RNA seq and Chip seq were employed to explore the underlying molecular mechanisms. The following hypothesis is proposed: The transcription factor Barx1 binds directly to the Dlx5 promoter and regulates the transcription of Dlx5, mediating the role of PDA in promoting cementum formation. The current research was designed for this purpose. The content of the research was listed as follow: Firstly, micro-CT and immunohistochemical staining were used to identify the effects of PDA on cementum formation in the orthodontic tooth movement model, and the expression of Barx1 and Dlx5 was detected. Then the expression of Barx1 in cementoblasts was up-regulated and down-regulated separately, to further clarify the role of Barx1 in cementoblasts. Finally, the exact binding site between Barx1 and Dlx5 were identified by means of Chip qPCR, fluorescein reporter assay and EMSA. Through the performance of this project, it is expected to reveal the molecular mechanism of PDA to promote orthodontic cementum regeneration, and provide new strategy for improving adult orthodontic effects.

开发有效的促进牙骨质再生策略对于成人正畸治疗具有重要临床意义。申请者前期发现聚多巴胺（PDA）能够有效促进牙骨质再生，促进成牙骨质细胞的分化，并结合RNA seq和Chip seq等高通量方法发掘了潜在的分子机制，提出了如下假说：转录因子Barx1能直接与Dlx5启动子结合并调控Dlx5的转录，介导了PDA促进牙骨质再生的作用。为此设计了本研究，内容如下：首先借助Micro-CT、免疫组化染色等方法，在大鼠正畸牙移动模型中明确PDA对牙骨质再生的作用特点，并检测Barx1、Dlx5的表达趋势；其次分别上调和下调成牙骨质细胞中Barx1表达，进一步明确Barx1在成牙骨质细胞中的作用表型；最后借助Chip qPCR、荧光酶素报告实验、EMSA等手段，明确Barx1与Dlx5启动子结合的准确位点。通过本项目的开展，有望揭示PDA促进正畸牙骨质再生的分子机制，为提升成人正畸效果提供新的思路。

牙骨质的再生和修复是牙周组织再生中的重要部分，关系到牙周附着的有效构建，探究其背后调控机制，并以此为靶点开发新型的材料具有重要意义。课题组在前期明确聚多巴胺（PDA）的牙周抗炎作用基础上，通过本项目进一步明确PDA对牙骨质代谢的调控作用和机制。首先，从细胞和分子层面明确了PDA在成牙骨质细胞矿化中的作用和基因变化，发现PDA能抑制成牙骨质细胞的矿化，并明确了PDA是通过进入细胞中影响基因表达发挥的作用。其次，探究了Barx1在成牙骨质细胞中的作用开展了研究，发现Barx1存在于成牙骨质细胞中，通过应用crispr方法构建了Barx1敲减细胞株，明确了Barx1的下调会导致成牙骨质细胞矿化减弱，而这种作用可能是通过wnt通路发挥作用的。最后，受PDA基材料仿酶活性的启发，课题组设计了多种新型无机杂化纳米材料，借助其仿酶活性实现体内外抗菌功能。此外，课题组基于上述研究工作，总结了以聚多巴胺为代表的微纳生物材料在口腔感染疾病中的应用现状，并提出了面临的挑战。通过三年的研究工作，项目组完成了预期研究内容，发表了SCI论文4篇，协助培养硕士生1名。