应力微环境下YAP/TAZ介导牙周膜肌成纤维细胞的分化机制

Periodontal ligament cells (PDLCs), as the senor and effecter of mechanical stimuli, change the phenotype and function during orthodontic tooth movement. This process is the key to mechanical signal transduction and integration. Considering the literature reviews and our previous research, we made a conclusion that differentiation from PDLCs to myofibrobalsts (MFB) under the mechanical stimulation might be regulated by RhoA/ROCK-F-actin-YAP/TAZ-TEAD signal pathway, in which YAP/TAZ played the central role. To test this hypothesis, we plan to trace the nucleocytoplasmic shuttling of YAP/TAZ. The YAP/TAZ-mediated PDLCs-myofibroblast transition under mechanical loading will be explored both in vivo and in vitro, through genetic manipulation of YAP/TAZ. Furthermore, the molecular mechanism of PDLCs-myofibroblast transition will be examined in detail in vitro, by medication or genetic manipulation of the signaling cascades in this signal pathway. This study is expected to uncover the mechanism of mechanotransduction in the periodontal ligament (PDL), and to provide new insights into biological strategies aiming at promoting the periodontal tissue remodeling and effectively controlling orthodontic tooth movement. This study also holds special hope for providing potential therapeutic targets for treatment of various kinds of myofibroblast-related diseases.

正畸牙移动过程中，牙周膜细胞作为牙周组织对力学刺激的感知器和效应器，会发生表型和功能的改变，此过程是力学信号得以传递和整合的关键步骤。结合已有研究线索和前期研究结果，我们认为力学刺激作用下牙周膜细胞向肌成纤维细胞的分化，很可能是由转录激活因子YAP/TAZ介导的力学-化学信号转导和整合，并通过RhoA/ROCK-F-actin-YAP/TAZ-TEAD信号通路实现的。为验证该假说，本研究以YAP/TAZ核浆穿梭为切入点，联合体内体外实验通过改变YAP/TAZ的表达，结合干扰RhoA/ROCK-F-actin-YAP/TAZ-TEAD信号通路上各不同位点，探索YAP/TAZ调控牙周膜肌成纤维细胞分化的分子机制。该研究将深入揭示力学信号在牙周膜中的传递过程，为正畸临床提高牙周组织改建水平、有效控制牙移动提供新的思路；同时为肌成纤维细胞相关疾病的治疗提供潜在的生物学靶点。

项目背景.正畸牙移动是在机械刺激触发下牙-牙槽骨复合体发生信号转导、细胞反应和组织改建的过程。牙周组织改建水平决定了牙移动的速率和矫治效果的稳定。牙周膜作为正畸力影响牙槽骨改建的中间“介质”，是参与其中的关键组织结构。然而对于力学刺激在牙周膜中的信号转导机制却鲜见突破。课题组前期发现，应力作用下，牙周膜细胞（periodontal ligament cells, PDLCs）向肌成纤维细胞(Myofibroblasts, Mfbs)的分化在应力诱导的牙周改建中发挥重要作用。本研究在此基础上进行延续，探讨了应力感应信号蛋白Yes相关蛋白（Yes-associated protein,YAP）在其中的调控及相关机制。.主要研究内容.1）构建大鼠正畸牙移动模型,收集不同牙移动阶段颌骨标本,检测牙移动效率及牙周膜中YAP的表达和Mfbs的分化；.2）体外培养人PDLCs原代细胞,施加不同力值和时长张/压应力,外源性添加RhoA/ROCK通路抑制剂,结合免疫荧光染色、RT-qPCR、Western Blot等检测手段,探索机械应力加载刺激下，YAP通过RhoA/ROCK通路,参与牙周膜中Mfb分化调控的级联通路；.3）构建含目的基因YAP的过表达慢病毒载体,结合RhoA/ROCK通路抑制剂,验证YAP调控牙周膜Mfb分化的分子机制。.重要结果及关键数据.1）在牙移动的不同阶段伴随YAP表达变化,表现为牙周膜张力侧YAP表达随牙移动加力时间增加而变化,其表达量与Mfb特异性标记物α–SMA的表达呈一定的正相关关系；.2）无论是张应力还是压应力均能刺激PDLCs向Mfbs分化,外源性添加RhoA/ROCK通路抑制剂能减弱张应力刺激诱导的Mfb分化,这一过程中YAP的mRNA水平及蛋白水平表达均降低；.3）过表达YAP能显著促进PDLCs向Mfbs分化,伴随RhoA/ROCK通路的激活以及TGF-β1的表达上调,在此基础上添RhoA/ROCK通路抑制剂则降低信号级联通路关键因子的表达活性，同时阻碍Mfbs分化。.科学意义.本研究揭示应力微环境中牙周膜细胞向肌成纤维细胞分化的分子机理，为研究力学信号在牙周膜中的传递和整合过程提供新的实验依据和治疗靶点。同时也为肌成纤维细胞相关疾病（如组织器官纤维化、肿瘤发展、瘢痕挛缩等）的治疗提供潜在的研究参考。