牙周病进程中炎性环境导致骨形成与骨吸收失衡的分子生物学机制研究

During the pathological processes of periodontitis, the inflammatory reactions are believed to accelerate alveolar bone loss via negative effects on bone remodeling. To understand the underlying mechanisms, researchers have investigated the role of inflammatory molecules in bone formation by osteoblasts or in bone resorption by osteoclasts. However, currently detailed information is still unavailable regarding the possible effects of the inflammatory reactions on the molecular signaling network responsible for the inter-relationship between osteoblasts and osteoclasts. In this grant application, we proposed to investigate the effect of an inflammatory micro-environment on two of the most important osteoblast-osteoclast signaling pathways: RANKL-OPG and ephrinB2-EphB4 pathways. Firstly, we will investigate the molecular mechanisms underlying the regulatory effects of the inflammatory molecules on the expression levels of the main components of the signaling pathways. Secondly, we will re-establish normal bone remodeling processes in an animal model via the adjustment of the RANKL/OPG ratio. Thirdly, we propose to investigate the changes in the regulatory effects of the ephrinB2-EphB4 pathway on bone remodeling via the establishment of an inflammatory environment in conditional gene knockout animal models. These proposed studies will shed a great deal of light on the mechanisms underlying the regulatory roles of inflammatory molecules in bone remodeling. The results derived from the proposed studies will also provide new targets for the development of novel periodontal therapies and therefore have great clinical significance.

在牙周病进程中，宿主炎症反应可打破骨组织内稳态，加速牙槽骨丧失。关于其作用机制，现有研究大多将成骨细胞（骨形成）或破骨细胞（骨吸收）作为独立个体分离开进行研究，因而缺乏炎性环境下成骨细胞和破骨细胞在信息交流和互相调控等方面是否存在失衡的准确信息。本课题组拟在前期研究的基础上，以成骨细胞和破骨细胞实现信息交换的两大分子信号网络（RANKL-OPG和ephrinB2-EphB4）为研究对象，通过体外实验检测炎性因子调控这两大分子信号网络表达水平的分子生物学机制。同时用体内试验探讨炎性刺激下，通过调整RANKL/OPG比例干预骨改建过程的可行性。另外，本项目将利用条件性基因敲除小鼠监控炎性环境下成骨细胞和破骨细胞间缺失ephrinB2-EphB4网络对骨改建的影响。本项目将使研究者更好地了解炎性因子对骨改建的影响，并为牙周病新药及新治疗措施的研发提供新的作用靶点，因而具有重要的临床和科研意义。

牙周炎是一种慢性、细菌感染性、炎症性疾病，其主要临床表现为牙齿软、硬支持组织的丧失。TNF-α作为一种重要的炎性因子，在牙周炎进程中起重要作用。本研究探讨了不同浓度的TNF-α 对小鼠前成骨细胞系MC3T3-E1成骨分化能力和EphB4信号表达的影响，并且研究了EphB4信号在TNF-α调控的成骨细胞成骨分化中的作用。同时，还初步探讨TNF-α调控EphB4信号的机制。另外，我们建立了小鼠下颌骨缺损模型，在骨缺损处应用带有EphB4和ephrinB2干扰载体的慢病毒实现相应信号的抑制，同时，对小鼠腹腔注射TNF-α创造全身炎性环境，评价全身炎症环境下EphB4和ephrinB2信号对小鼠下颌骨缺损愈合的影响，揭示了EphB4信号在调控炎症微环境下骨形成中的重要作用。此外，我们还建立了牙周炎大鼠下颌骨缺损模型，进一步评价局部牙周炎症环境对下颌骨缺损愈合的影响并进一步探讨ephrinB2-EphB4信号的作用。实验的重要结果：1）小剂量TNF-α通过激活EphB4信号促进成骨，而高剂量TNF-α通过抑制EphB4信号抑制成骨，其机制有待进一步研究。2）高剂量TNF-α通过激活NF-kB，进而抑制EphB4信号。3）大鼠实验性牙周炎可抑制颌骨骨缺损的愈合，同时抑制EphB4信号的表达。4）系统性炎症状态下，EphB4信号同样显示出促骨再生的作用。5）TNF- α对Runx2下游9.0kb BSP 启动子的激活有抑制作用。上述结果均为国内外首次发现，揭示了炎症环境导致骨吸收的新机制，为炎症环境下骨吸收的预防和治疗提供新思路。