机械压力信号在人牙龈成纤维细胞纤维化传导通路中的初步研究

Gingivae is an important part of periodontium.Some clinical observations and basic researches revealed that if the gingivae was compressed during orthodontic treatment,it would gain increased elastic properties and the fibers in it were tracted and the volume of elastic fiber was increased.It was believed that these changes of the compressed gingivae would play an important role in relapse. Therefore, on the regard of preventing relapse and improving orthodontic treatment efficiency，it is of great significance to investigate the orthodontic gingival remodelling mechanism in-depth.Preliminary studies have shown that mechanical force will break the balance between human gingival fibroblasts（HGFs） synthesize and degrade collagen, suggesting that increasing collagen synthesis but reducing degradation will lead to extracellular matrix deposited. In addition, this pathological process is similar to the fibrosis. So, it can be speculate that the mechanical force translate into biological signals activate HGFs that regulate the metabolic activity of collagen by the pathway of fibrosis, and then participate in the gingival tissue reconstruction. To certify this hypothesis, we will use HGFs and PLGA-collagen composite biological scaffold materials to build the three-dimensional cell model and carry out the mechanical loading by strain loading apparatus. Analysising by proteomics and bioinformatics, the expression of proteins before and after loading are examined in the overall, finally the proteins associated with the pathway of fibrosis will be filtered out. And, by classical methods of cell signal pathway, such as relative gene enhancements, silencing and blockade, our study is to explore the mechanism of key proteins in the pathway of fibrosis and trying to illuminate how mechanical force signal regulate the remodel of gingival tissue through and pathway of fibrosis. So it is necessary to study the remodeling reaction of gingival tissue in variety of mechanical environment and this will provide theoretic guidance to the clinical treatment.

牙龈是牙周组织的重要组成部分，临床观察和一些基础研究认为，正畸治疗过程中，牙龈受压堆积导致的弹性性能增加、纤维受牵拉和弹性纤维量增加是导致复发的原因之一，因此，研究牙龈的改建，对有效防止复发和提高矫治效率具有重要意义。在前期研究发现，机械力打破了人牙龈成纤维细胞（HGFs）合成与降解胶原之间的平衡：胶原合成增加，而降解减少，细胞外基质沉积。这与纤维化改变有着相似的病理过程。我们推测，机械力信号通过纤维化的传导通路调节HGFs胶原纤维代谢活动，从而参与牙龈组织改建。为证实这一假说，我们将构建HGFs三维细胞重力加载模型，采用蛋白质组学方法，筛选出与纤维化传导通路相关蛋白；并利用相关基因增强、沉默及阻断等研究细胞传导通路的方法，探究关键蛋白在其中的作用机制，试图阐明机械力信号如何通过纤维化传导通路调控牙龈组织改建过程，以期丰富牙移动机理，为指导临床、提高疗效和稳定性等方面提供坚实的依据。

正畸治疗中牙龈的变化直接影响矫治效率及矫治后稳定性，其机制尚未阐明。在前期研究发现，静压力刺激HGFs胶原合成增加，而降解减少，细胞外基质沉积。这与纤维化改变有着相似的病理过程。推测，机械力信号激活HGFs，通过纤维化的传导通路调节胶原纤维代谢活动，从而参与牙龈组织改建。为证实这一假说，我们成功构建了PLGA-胶原复合生物支架材料与HGFs三维细胞重力加载模型，摸索出加载HGFs三维培养复合体的最适力值为25g/cm2。采用蛋白质组学技术，运用生物信息学分析方法，整体地观察HGFs加力和未加力组蛋白表达谱的差异，筛选出主要参与HGFs信号传导并处在关键节点位置的10种蛋白。进一步研究发现1.静压力可以刺激HGFs高表达CRT和TGF-β1、COL-Ⅰ；CRT对TGF-β1的合成有重要作用；TGF-β1对COL-Ⅰ的合成有重要作用。2.静压力刺激作用下，HGFs整合素α5β1感应力学信号，调控下游FAK的表达和磷酸化，影响细胞合成COL-Ⅰ。3.静压力下，过表达Smad4能抑制HGFs增殖。过表达Smad4可以调节HGFs中Caspase-3和Bcl-2的表达。即Smad4可以促进Caspase-3的表达、抑制Bcl-2的表达。我们认为，静压力刺激HGFs，细胞膜上整合素α5β1感应力学信号传至细胞骨架，内质网发生应激反应，CRT的表达量增多，刺激TGF-β1高表达，激活TGF-β1致纤维化的相关信号通路，导致COL-Ⅰ合成增多，MMP-1合成减少，造成细胞外基质沉积，发生牙龈纤维化改变。本研究为机械力信号如何调控牙龈组织改建过程提供了更深入的见解，这可能有助于提高正畸矫治效率和治疗后的稳定性。