根尖周炎中Dickkopf-1（Dkk-1）诱发骨组织去偶联的机制

Physiologically, an episode of bone resorption is followed by an equivalent amount of bone formation, which is called coupling. However, the presence of inflammation in periapical tissues hampers bone formation and causes the uncoupling from bone resorption to formation, which would lead to bone loss in periapical area. Hence, it is important to restore the coupling process to promote bone formation in the treatment of apical periodontitis. This study aims to elucidate the mechanism of Dkk-1-mediated bone uncoupling in apical periodontitis. First, osteogenesis will be induced in bone marrow stromal cells (BMSCs). In order to explore the effect of inflammation on osteogenesis, the cells will be treated with Pg. LPS at different stages of osteogenic differentiation. Then Dkk-1 and Wnt signal pathway will be studied. The role of inflammatory cytokines and miRNA which may be involved in this process will be investigated. Second, BMSC will be treated with Pg. LPS and Dkk-1 recombinant protein at different stages of osteogenic differentiation. Then the markers of osteogenic differentiation will be measured to illustrate the mechanism how inflammation or Dkk-1 interfere coupling process from bone resorption to bone formation. Finally, experimental apical periodontitis will be established on rats. Then dynamic bone histomorphometry will be performed on the alveolar bone. The gene and protein expression of osteogenic markers and Wnt signal will be investigated on the bone samples. And bone loss will be compared between normal rats and those treated with Dkk-1 inhibitor. In a word, this study will elucidate the mechanism of Dkk-1-mediated bone uncoupling in apical periodontitis and try to find the new therapeutic target.

慢性根尖周炎是一种炎性骨组织疾病。炎症破坏骨吸收与骨形成之间的平衡，引起骨组织去偶联，导致根尖周骨质丧失，因此恢复骨偶联、促进骨形成是治疗根尖周病、逆转根尖周骨质丧失的关键。本项目立足于炎性环境中骨组织修复重建这一主题，分别从体外细胞实验、动物疾病模型两方面进行研究，探讨炎症对成骨细胞分化的影响，分析炎症影响骨形成的具体环节和靶点，揭示Dkk-1-Wnt信号在根尖周炎中诱发骨组织去偶联的机制，探索使用Dkk-1抑制剂恢复骨偶联、促进骨形成、逆转牙槽骨吸收的可能性，为根尖周病的治疗提供新的思路和方法。

生理条件下，骨吸收和骨破坏间存在平衡，骨吸收的同时伴随有等量的新骨形成，从而保持骨量的不变，此平衡被称作骨偶联。在骨偶联中，各种因子扮演着骨偶联所需要的信号从而维持骨代谢平衡。当外界微环境发生改变时，骨去偶联信号引发骨吸收和骨形成失平衡，从而表现出骨丧失或骨异常增生。慢性根尖周炎常常伴随有根尖牙槽骨的丧失。当根尖周组织遭受病原微生物侵袭时，局部将发生炎症反应，活化破骨细胞，引发骨吸收，抑制新骨形成，最终导致根尖周牙槽骨丧失。从这一点来讲，根尖周炎是一种典型的骨去偶联疾病，而局部炎症反应正是引起骨去偶联的病因，然而这其中的具体分子机制尚未明确。Dkk-1是一种内生性分泌蛋白，作为Wnt信号通路的抑制剂可负向调节经典的Wnt信号通路，并抑制成骨分化及骨形成，已有研究表明，类风湿性关节炎患者血清中的Dkk-1水平明显高于正常人，而一些前促炎因子如IL-1β和TNF-α也可体内和体外促进Dkk-1的表达。由此，可以推测Dkk-1可能在炎性条件下作为去偶联信号分子调节经典的Wnt信号通路从而引起骨丧失。.本研究将分离培养大鼠骨髓间充质干细胞（BMSC）及小鼠胚胎成骨细胞前体细胞MC3T3-E1细胞，使用由大肠杆菌提取的脂多糖（Ecoli. LPS）、Dkk-1重组蛋白、Dkk-1siRNA等刺激细胞，研究炎症对大鼠骨髓间充质干细胞增殖分化的影响，探讨炎症条件下Dkk-1在成骨分化其中的作用。结果表明E.coli. LPS刺激下的MC3T3-E1细胞成骨分化前中后期标志物Runx2、Osx、BSP mRNA表达下降，碱性磷酸酶ALP活性下降，并观察到Wnt/β-catenin信号通路的抑制；与此同时炎症条件下Dkk-1的蛋白表达水平也在成骨分化过程中发生相应改变，而Dkk-1抑制剂可减轻大鼠根尖周炎模型中的骨质丧失。.本研究立足于根尖周骨组织修复重建，围绕炎症反应诱发骨组织去偶联的关键分子Dkk-1，研究炎症反应对Dkk-1的调控作用，探讨从间充质干细胞到前成骨细胞，从成骨细胞到骨形成，炎症对成骨分化不同环节的影响，分析Dkk-1-Wnt信号在其中的调节作用，为治疗根尖周炎，逆转牙槽骨吸收，恢复骨偶联，促进骨形成提供理论依据。