Vangl调控破骨细胞活化的机制研究

Periapical periodontitis is a periapical disease characterized by bone resorption. Osteoclast activation, which is characterized by its polarization and enzyme secretion, is the key point in periapical bone resorption. However, the molecular mechanism of osteoclast activation has not been fully understood. Our previous study observed that Vangl was asymmetrically enriched in the side of active osteoclast membrane contacting bone surface; Vangl-knock-out in osteoclast resulted in elevated bone density, implying Vangl plays an important role in osteoclast activation. Therefore, we assumed that Vangl regulates osteoclast activation by affecting its polarization and relative enzymes. To test our hypothesis, we generated HA-Vangl2 transgenic mice to observe the relationship between Vangl and osteoclast activation markers; we established CathCre+; Vangl1-/-; Vangl2c/c transgenic mice to remove Vangl in osteoclast, and will examine the changes in osteoclast polarization and the function of the enzymes by immune fluorescence， scanning electron microscope, ELISA and pit assay, et al.; Western blot and other relevant methods will be also used to understand if Vangl acts through p38、RhoA、JNK. The molecular mechanism will be investigated by microarray analysis as well. As the high prevalence of periapical periodontitis, our study will expand our understanding of molecular mechanism of osteoclast activation, which will provide new therapeutic targets for the disease.

根尖周炎是以骨吸收为特征的疾病，以极性形成及酶分泌为特点的破骨细胞活化是骨吸收的关键环节，目前其机制尚未完全明确。我们前期研究发现，Vangl在活化的破骨细胞中极性表达于骨吸收侧；破骨细胞敲除Vangl可致小鼠骨密度增加；提示Vangl参与调解破骨细胞活化。我们推测Vangl可能通过影响破骨细胞极性及相关酶调节其活化。本项目拟通过建立 HA-Vangl2转基因小鼠观察Vangl与破骨细胞活化标志物的关系；建立CathCre+;Vangl1-/-;Vangl2c/c转基因小鼠，采用免疫荧光、扫描电镜、ELISA、Pit assay等技术检测Vangl对破骨细胞极性形态结构及破骨相关酶功能的影响；运用表达谱芯片筛选Vangl调节破骨细胞活化的新靶点；通过Western Blot等技术证明Vangl对p38、JNK、RhoA的作用，以期阐明破骨细胞活化的分子调节机制，为根尖周炎治疗提供新思路。

根尖周炎是口腔常见病与多发病，传统治疗方式无法治疗骨质缺损，影响患者治疗后生活治疗，目前其机制尚未完全明确。在本项目资助下，我们通过建立条件性敲除Vangl基因小鼠，发现破骨细胞敲除Vangl可致小鼠骨密度增加，提示Vangl参与调解破骨细胞活化；通过建立 HA-Vangl2转基因小鼠，检测Vangl在破骨细胞与骨接触侧极性表达，在体外破骨诱导过程中表达增加；Vangl表达与肌动蛋白环表达重合，通过pit assay检测，发现Vangl敲除后，骨吸收能力降低，提示Vangl参与破骨细胞的骨吸收过程。通过成骨细胞及骨细胞的免疫组化、HE染色、qPCR检测等相关基因检测，发现骨量增多导致骨细胞的产生的Sost分泌增加，经典 Wnt信号通路被抑制，进而成骨向分化降低。本研究初步阐明了Vangl调控破骨细胞活化的分子机制，为根尖周炎及骨质疏松等疾病的治疗提供了理论基础和新的思路。