miR-26a介导的正畸力调控骨髓间充质干细胞成骨分化机制研究

Orthodontic force modulates osteogenic differentiation of oralfacial bone marrow mesenchymal stem cell（OBMSCs）to couple bone remodeling in orthodontic teeth movement. MicroRNAs are key regulators of stem cells differentiation and cell fate determination. Whether orthodontic force modulates osteogenic differentiation of OBMSCs by regulating microRNAs expression is unknown. Our group have found that miR-26a regulates osteogenic differentiation of BMSCs derived from long bone through Wnt/β-Catenin and BMP signaling pathway. Moreover, the expression of miR-26a is modulated by mechanical force, suggesting that orthodontic force modulates osteogenic differentiation of OBMSCs by regulating miR-26a expression. In this study, we will take advantage of mechanical force delivery system, OTM animal model, bioinformatics, and gain- and loss-of-function analysis to confirm the role of miR-26a in OBMSCs osteogenic differentiation, investigate whether orthodontic force regulates miR-26a expression to control osteogenic differentiation of OBMSCs, and the importance of this miRNA-based mechanism in orthodontic teeth movement. This study will reveal for the first time the microRNA-mediated mechanism in bone formation during orthodontic teeth movement and unclose the basic molecular mechanism, and also provide novel strategy to improve bone reconstruction during orthodontic treatment.

正畸力通过调控颌骨骨髓间充质干细胞（OBMSCs）的成骨分化参与正畸骨塑建过程，而microRNAs是干细胞分化的重要调节子。正畸力是否通过调控microRNAs影响OBMSCs的成骨分化目前并不清楚。本课题组前期体内外研究结果已表明miR-26a通过调控Wnt和BMP信号通路影响长骨BMSCs的成骨分化；体外应力可调控BMSCs miR-26a的表达水平，提示正畸应力通过改变miR-26a的表达调控OBMSCs成骨分化。本研究拟借助细胞应力加载模型及大鼠正畸牙齿移动模型，采用生物信息学、获得性和缺失性功能分析等方法，检测miR-26a在OBMSCs成骨分化中的作用及分子机制，明确正畸应力通过调控miR-26a表达影响OBMSCs分化，探讨该调控机制在正畸骨塑建中的作用。本课题将首次从转录后调控水平加深对正畸力作用分子机制的认识，并为探索改善正畸治疗的新策略提供实验基础。

正畸力通过调控间充质干细胞成骨分化参与牙周骨塑建，microRNAs是重要的干细胞分化调节子。本课题通过构建微尺度非接触拉伸水凝胶的三维应变微环境模型，发现miR-26a、miR-200a/b/c簇、miR-10b、miR-202均为重要的力应答miRNAs，通过Smad1/5/9、GSK-3β、Smurf1等作为靶分子分别介导TGF-β/BMP、Wnt、PI3K信号通路调控干细胞成骨分化；力应答miRNAs表达变化与衰老颌骨骨髓间充质干细胞（OBMSCs）成骨分化下降相关。进一步将富含miRNAs和蛋白质的骨诱导基质小泡（Exosomes）作为研究目标，体外通过三维应力微环境调控健康组织来源牙周膜干细胞的exosomes及其miRNAs内容物变化，对提取的SM-Exo形态、粒径分布、标记蛋白表达、分泌量的差异等外泌体固有特征以及与SM-Exo共培养的OBMSCs增殖、迁移、凋亡和成骨分化改变，确定适宜的促增殖迁移并成骨分化和凋亡保护的机械微环境条件。同时结合基质小泡内miRNA-seq分析，进一步筛选确定miR-200a/b/c簇、miR-10b为重要调节因子。构建SD大鼠炎症和非炎症性牙槽骨缺损模型，分别将PBS、Matrigel、Exo-Matrigel、SM-Exo-Matrigel注射到不规则形态牙槽骨缺损内，由Micro-CT、H&E及Masson染色明确其促牙周骨再生的能力，结合免疫组化和免疫荧光染色从体内进一步阐明Smad1/5/9、GSK-3 β、Smurf1作为靶分子参与TGF-β/BMP、Wnt、PIk/Akt等相关信号调控的具体机制。再利用支架材料负载miR-200a/b/c簇、miR-10b的模拟物mimic和抑制剂inhibitor从体内验证其促牙槽骨缺损修复能力。本课题将为探索提升牙周病正畸治疗疗效的新策略提供实验基础。