正畸压应力通过Ets-1/Tks5信号通路调控侵袭性伪足促进破骨细胞形成的机制研究

Accelerating orthodontic tooth movement can significantly reduce treatment duration and risks of side effects such as root resorption and open gingival embrasure spaces. The rate-limiting step in orthodontic tooth movement is considered to be the formation of osteoclasts and its subsequent bone resorption at the compression side. Osteoclasts are multinucleated giant cells created by cell-cell fusion of monocyte/macrophage precursors. Recent studies have shown that circumferential invadopodia are important for cell-cell fusion. However, the role and mechanisms underlying the regulation of circumferential invadopodia in osteoclast precursors under orthodontic compressive force remain unexplored. Our preliminary experiments found that the compressive force loading on RAW 264.7 macrophages resulted in a significant increase of circumferential invadopodium formation and cell-cell fusion. Moreover, tyrosine kinase substrate with five SH3 domains (Tks5), which is recognized as a “molecular switch” that directs invadopodium formation, and E26 transformation-specific-1 (Ets-1) was upregulated in the mechanical loading cells. Bioinformatics revealed that there is a sequence-specific Ets-1 binding site in the Tks5 promoter. Knockdown of Ets-1 suppressed the compressive force-induced circumferential invadopodium formation and cell-cell fusion, as well as Tks5 expression. Interestingly, a recent study reported that Ets-1 can bind Integrin β1 promoter and promote its expression. Integrin β1 is important for Girdin activation, which in turn causes Tks5 activation. Therefore, we hypothesized that orthodontic compressive force modulates Ets-1/Tks5 pathway to promote the formation of circumferential invadopodia in osteoclast precursors, finally accelerate cell-cell fusion. To confirm our hypothesis, we will employ macrophage cell lines and rat model of orthodontic tooth movement as in vitro and in vivo models. The effects of compressive force on circumferential invadopodium formation and cell-cell fusion of osteoclast precursors will be determined by immunofluorescence confocal microscopy and TRAP assays, respectively. Mechanistically, the macrophages will be transfected with different constructs, such as expression vectors or shRNA. Then luciferase reporter assays, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) will be performed to confirm the interaction of Ets-1, Tks5, and Integrin β1 under compressive force. Finally, the Ets-1/Tks5 pathway will be examined in the Ets-1-knockout and wild-type rat model of the orthodontic tooth movement. It is expected that these experiments will at least partly elucidate the molecular mechanism by which compressive force promotes circumferential invadopodium formation and cell-cell fusion in osteoclast precursors and identify novel therapeutic targets for improving the quality and efficiency of orthodontic treatments.

随口腔医学发展，越来越多人接受错颌畸形正畸治疗。正畸疗效关键步骤之一是正畸应力如何得到响应，诱导破骨前体细胞融合分化为破骨细胞，从而实现正畸牙移动，但机制还不完全清楚。近来研究提示侵袭性伪足是促进破骨细胞形成的关键因素。申请人前期发现正畸应力可促进破骨前体细胞侵袭性伪足形成，其机制可能与Tks5蛋白活化有关。生物信息学分析发现Tks5启动子上存在Ets-1特异结合位点，敲低Ets-1表达可抑制正畸应力促进的侵袭性伪足形成和Tks5活化。此外，有报道Ets-1可激活Integrin β1表达，后者可能在Tks5活化过程中起重要作用。因此，该项目拟利用正畸应力加载仪及大鼠牙移动模型等，探索工作假说：正畸应力是否通过Ets-1和Integrin β1表达响应，诱导Tks5活化，促进侵袭性伪足形成，进而导致破骨细胞形成。预期研究结果对理解正畸治疗的细胞分子机制和潜在调控靶点具有重要科学意义。

目的：错颌畸形是一种常见病，我国发病率72.92%，其中复杂错颌畸形严重影响颜面美观、口腔功能对患者造成严重心理障碍；且矫治诊断设计复杂、疗程长，已成为口腔医学亟待攻克的科学问题。其发病和治疗与牙周、颅面、颞颌关节的发育与组织改建密切相关，是口腔临床亟待解决的热点难点问题。正畸疗效的关键之一在于正畸应力如何得到响应，从而诱导牙周膜干细胞成骨向分化，促使牙周组织改建，最终维持牙周稳态平衡。本研究旨在阐明正畸力学与化学刺激间的转化关系，构建稳定健康的“牙骨质-牙周膜-牙槽骨三明治样结构”，为临床诊断依据和治疗方法提供新思路。.方法：分离培养人来源的牙周膜干细胞，通过EdU实验、划痕实验和茜素红染色检测Girdin蛋白在受牵拉牙周膜干细胞增殖、迁移及成骨分化中的作用；分离巨噬细胞来源的外泌体，通过茜素红染色及成骨相关基因/蛋白的定量分析检测M0/M1/M2型巨噬细胞外泌体对牙周膜干细胞成骨向分化的作用；构建大鼠牙周组织缺损模型，评估巨噬细胞外泌体对缺损牙周组织的修复与再生作用。.结果：本研究发现Girdin蛋白在受牵拉牙周膜干细胞增殖及成骨分化过程中均发挥正向调控作用；此外，M2型巨噬细胞外泌体能明显促进牙周膜干细胞成骨向分化，最终促进缺损牙周组织再生。.结论：本研究证实Girdin在牙周膜干细胞牵张成骨过程中发挥关键作用，初步明确了巨噬细胞外泌体调控牙周膜干细胞再生牙周组织的可能性及相关机制，为正畸过程中维持牙周稳态提供新思路和理论支持。