核骨架蛋白在张应变调控人牙周膜细胞功能中的作用及其机制

Cyclic strain which promotes the proliferation and migration of human periodontal ligament cells in the orthodontic reconstruction of periodontal tissues plays an important role. After the mechanical force used in the periodontal ligament cells,the nuclear envelope protein as an important molecular mechanics sensitive protein can serve as the nucleus changes, to further interact with related transcription factors regulate gene transcription, participate in the induction of cell proliferation, migration cell activity, finally complete reconstruction of periodontal tissue. Human periodontal ligament cells was subjected to cyclic strain using a Flexercell Tension Plus system, the application of confocal laser and electron microscopy techniques to observe the distribution of periodontal ligament cells nuclear envelope proteins and morphology.At the same time with western blotting, flow cytometry, RNA interference, and construct high technology means such as expression vector plasmid transfection technique detection in periodic strain control nuclear envelope proteins and the related signal molecule expression changes, and its on cell proliferation, migration and other cellular activity function change. Using proteomics and chip technology can filter out nuclear envelope proteins directly in significant role of transcription factors, take the luciferase report gene experiment, yeast two hybrid test methods, such as further explore nuclear envelope protein with the relevant signal factor interaction mechanism and its regulation on related gene loci, clarify nuclear envelope proteins in the role of regulating the periodontal ligament cells, cyclic strain to reveal the nuclei of biological mechanics within the nuclear signal transduction mechanisms. Finally using animal models of orthodontic tooth movement in rats as the research object, from histological level to verify our experimental results, so as to further perfect our experiment conclusion. This study will help to further clarify orthodontic periodontal tissue reconstruction mechanics biological mechanism, research on mechanism of orthodontics treatment has theoretical and clinical significance.

应力作用下人牙周膜细胞增殖和迁移在正畸牙周组织重建中起重要作用。机械力作用于牙周膜细胞后，细胞核重要组成分子核骨架蛋白作为力学敏感蛋白发生改变，与相关转录因子作用调控基因转录，参与诱导细胞增殖、迁移活动，完成牙周组织的改建。本项目采用Flexcell细胞张应变加载体外培养人牙周膜细胞模型，激光共聚焦和电镜技术观察细胞核骨架分布及形态结构；免疫印迹、流式细胞术、RNA干扰和高表达载体质粒转染技术检测在周期性张应变调控牙周膜细胞中核骨架蛋白及相关信号分子的表达变化，核骨架蛋白对细胞活动功能变化的影响；蛋白组学及芯片技术筛选出和核骨架蛋白作用的蛋白和转录因子，采取荧光素酶报告基因实验、酵母双杂交实验探究核骨架蛋白与相关信号因子的作用机制及对相关基因的位点调控，阐明其在张应变调控中的作用，揭示细胞核对生物力学信号传导的核内机制，并用大鼠正畸牙齿移动动物模型为研究对象，从组织细胞水平验证机制。

正畸治疗是通过矫治器产生或传递应力作用于牙或颌骨，导致牙周组织或口颌系统相关的颌骨、软骨、肌肉发生重建，使牙或颌骨产生移动的过程。人牙周膜细胞作为牙周膜中的主体细胞，是口腔正畸矫治力的效应细胞之一。它能将力学载荷转化为分子信号，调节细胞功能，在机械应力诱导的正畸牙周组织重建中起重要作用。细胞骨架是位于细胞质内的蛋白纤维网架体系，最新文献报道，细胞骨架和细胞核之间存在机械连接，它作为细胞外基质—整合素—细胞骨架—细胞核网络系统的核心部分通过调节骨架的重排和细胞形态的改变，能够实现力学信号向生物化学信号的转导，这也提示机械力可贯穿细胞质到达细胞核细胞核是真核细胞进行各种生命活动的主要控制中心，其不仅是遗传物质储存、复制和转录的场所，还可以调节细胞的代谢、生长和分化等多种功能。细胞核骨架蛋白作为细胞核内骨架对胞核的支撑以及胞核的刚度有决定性的作用，而核骨架蛋白laminA和其剪接体laminC是力学敏感因子，当人牙周膜干细胞受到不同幅度的周期性张应变力学刺激时，laminA/C的表达水平会发生变化，并参与了人牙周膜干细胞功能状态的调控。在本项目中我们成功分离培养出原代人牙周膜干细胞并通过免疫组织化学和流式细胞术等实验鉴定其为间充质来源的细胞；应用Flexcell FT-5000周期性张应变加载系统对人牙周膜干细胞进行力学加载，验证了生理性张应变和病理性张应变条件对人牙周膜干细胞成骨向分化的影响及人牙周膜干细胞中的LaminA/C表达量的变化；成功验证了LaminA/C表达量的改变对人牙周膜干细胞增殖，迁移及成骨向分化的影响；在动物实验中成功构建了大鼠正畸牙齿移动动物模型观察牙周组织改建情况及LaminA/C在牙周组织的表达变化情况。通过本项目的研究，阐明 LaminA/C 在周期性张应变作用下，影响人牙周膜干细胞的增殖和迁移功能，明确 LaminA/C 作为核内力学敏感蛋白在正畸牙周组织改建中的重要作用，揭示力学信号传导的核内机制及其与胞浆内信号传导通路之间的相互联系，为牙周研究提供力学生物学的新思路。