STAT3信号在正畸应力调控骨质疏松牙槽骨成骨改建中的作用及机制研究

Mechanical stress of orthodontic treatment could accelerate the orthodontic tooth movement (OTM) in the ovariectomied (OVX) rats，which also impacted osteoporosis in the alveolar bone. Previous studies paid more attention to the osteoclasts and bone resorption instead of osteoblasts and its function. However, our preliminary work indicated the promoted alveolar osteogenesis display a pivotal role in bone remodeling. The mechanism how mechanical stress could transform to the biological signaling in the alveolar bone with osteoporosis and strengthen the bone formation remains unclear. The previous screening of Reverse Phase Protein Array (RPPA) assay and predicting of Ingenuity Pathway Analysis (IPA) employed in our study indicated the Signal Transducer and Activator of Transcription 3(STAT3) could be a key factor potentially in the alveolar bone remodeling. With a review of the literature and preliminary data, we hypothesized that the activation of STAT3 by mechanical stress could prompt the osteogenic differentiation of BMSCs, which leads to the bone formation of alveolar bone with osteoporosis. This present study will employ molecular biological techniques including the chromatin immunoprecipitation assay (ChIP), small RNA interference, exogenous overexpression of targeted gene and so on to explore the underlying mechanisms how STAT3 regulate the downstream proteins, such as Runx2, Osterix, etc. Meanwhile, we are going to investigate the mechanosensitive signaling pathways through activating or inhibiting the upstream pathway. Finally, the validation of in vivo will be done by measurement of employing the histomorphometry, sequential fluorochrome labeling and micro-CT to investigate the function of osteoblasts and osteogenesis during the OTM in ovariectomied rats transfected with lentivirus. This work will systematically study the roles of STAT3 in the bone remodeling of osteoporosis regulated by mechanical stress of orthodontic treatment, which could develop a new sight of mechanical biology in the bone remodeling during the fast teeth movement and the new target improving the bone formation.

正畸力在骨质疏松的牙槽骨中使牙移动速度加快，以往研究聚焦在破骨吸收方面，而成骨方面研究少见，我们前期发现成骨改建增强在这一过程中发挥了保驾护航的关键作用，但力学信号如何引发骨质疏松牙槽骨内生物学信号、启动成骨向改建的调控机制有待阐明。课题组通过RPPA反相蛋白芯片筛选及IPA预测，发现信号转导和转录活化因子3(STAT3)信号可能起重要调控作用，通过前期实验初步验证并结合文献，提出假设：应力可能通过激活STAT3信号促进BMSCs成骨向分化，从而在增强骨质疏松牙槽骨的成骨向改建中发挥重要作用，但具体机制不清。本项目拟在前期基础上，结合细胞培养和动物模型，利用免疫共沉淀、基因沉默或过表达、激活或阻断上游通路等分子生物学技术，并进行体内验证，系统研究STAT3信号在力调控骨质疏松牙槽骨成骨改建中的机制，既为骨质疏松牙移动的骨改建机理提出新认识，又可发现新靶点调控骨形成，开拓新的力学生物学思路。

骨质疏松状态下正畸牙移动速度加快。以往研究聚焦在骨质疏松状态下破骨活性增强导致牙移动速度加快，而成骨方面的研究少见。课题组前期发现力学促进的成骨改建增强在维护骨质疏松牙移动安全性方面发挥了保驾护航的关键作用，并且通过体外应力加载系统发现信号转导和转录活化因子3 (STAT3)信号可能在应力调控成骨分化中发挥关键作用，但具体调控机制不清。.课题组通过建立体外应力加载模型及正畸牙移动模型，发现STAT3信号抑制剂AG490不但可在体外阻断力学介导的成骨分化，也可在体内降低正畸牙移动速度并导致牙槽骨骨量丢失。在原计划基础上，通过构建条件性敲除STAT3小鼠（Stat3Osx），发现：①成骨细胞内STAT3信号失活可通过降低成骨改建活性导致小鼠发生骨质疏松症; ②通过RNA-seq、Chip、Luciferase等分子生物学技术，发现STAT3可通过结合在Ocn、Dlx5等关键成骨基因的启动子区域调控其表达；③STAT3与RUNX2协同调控下游基因表达，进而促进成骨改建；④构建诱导性条件敲除小鼠（Stat3Col1-ERT2）并在此基础上构建正畸牙移动模型，发现成骨细胞STAT3失活可降低小鼠正畸牙移动速度，且使得牙槽骨中成骨细胞数量及活性均被抑制进而导致牙槽骨骨质疏松。综上，本项目阐明了在正畸牙移动（尤其是骨质疏松状态下）过程中，应力可通过STAT3直接调控Dlx5、Ocn等关键成骨基因的表达，促进牙槽骨的成骨改建，从而在骨稳态的维持及应力调控中均发挥重要作用，具有潜在的临床价值，同时完善了正畸理论和相关骨生物力学理论。.在运用以上条件性敲除小鼠运基础上，我们发现成骨细胞及其前体细胞可分为不同功能亚群，然而力学响应的成骨细胞亚群尚不明确，这为进一步研究奠定了基础并指明了方向。.课题已顺利完成考核目标：①由本基金所资助的直接相关文章已发表16篇（其中SCI 收录论文已发表8篇，中文核心期刊8篇），已录用2篇（其中SCI一篇，中文核心1篇）。②会议发言：国际会议壁报展示2次，国内会议发言5次，壁报展示3次。③已培养硕士研究生2名，在读6名。④课题负责人晋升主任医师和博导研究生导师。