颅骨锁骨发育不全致病基因RUNX2通过调控miR-31介导的牙槽骨改建过程影响牙齿替换的机制研究

Delayed or failed eruption of the permanent successors is a typical feature of cleidocranial dysplasia, which seriously affects the quality of the patients’ life. However, the underlying mechanism is still unclear. In previous study, we found that RUNX2 mutation, the pathogenic gene of cleidocranial dysplasia, down-regulated the osteogenic potential of odontogenic cells and its ability to support osteoclast differentiation and also the expression of miR-31, which regulates the differentiation of osteoblast and osteoclast. Therefore, the hypothesis is raised that RUNX2 mutations disturb the alveolar bone remodeling during tooth replacement by regulating miR-31. In order to test this hypothesis, the effect of RUNX2 mutation on the differentiation of osteoblasts and osteoclasts by dental follicle cells, pre-osteoblast and pre-osteoclast will be first investigated. Thereafter, the mechanism of RUNX2 regulating bone remodeling via miR-31 will be explored by luciferase reporter assay, ChIP assay, lentivirus transfection, Rescue assay, miR-31 target gene prediction and validation. Lastly, the bone formation and bone resorption of the tooth root slice combined with scaffold material and pre-osteoblasts or pre-osteoclasts transfected with RUNX2 mutant was explored in nude mice in order to verify the in vitro findings. This study explores the mechanism that underlies impaired tooth replacement in cleidocranial dysplasia.

颅骨锁骨发育不全综合征患者出现牙齿替换障碍，严重影响患者生活质量，但其机制仍未阐明。我们在前期工作中发现，其致病基因RUNX2突变下调牙源性细胞的成骨向分化能力和诱导破骨细胞分化的能力，并影响miR-31表达水平，而miR-31可调控成骨细胞和破骨细胞分化。因此，我们推测RUNX2突变通过调控miR-31干扰成骨细胞和破骨细胞分化，导致牙槽骨改建异常。针对此假说，我们将深入研究RUNX2突变对牙囊细胞诱导成骨细胞和破骨细胞分化能力的影响。其次，通过荧光素酶报告基因实验、染色质免疫共沉淀、慢病毒转染、“拯救”实验、miR-31靶基因预测和验证等方法，逐级分析RUNX2突变通过调控miR-31影响牙槽骨改建的机制。最后，通过观察吸附有RUNX2突变体细胞的牙根片段在裸鼠体内的骨形成和骨吸收情况，体内验证miR-31在RUNX2介导的牙槽骨改建中的作用，以揭示该综合征患者牙齿替换障碍的发病机制。

颅骨锁骨发育不全（Cleidocranial dysplasia, CCD）是一种罕见的常染色体单基因显性遗传病，致病基因为RUNX2。该疾病表现为全身多处骨骼发育不良和牙齿发育异常，其中口腔问题、尤其是牙齿替换障碍是影响此类患者生活质量和促使其求医的主要因素。因此，本课题旨在探讨 RUNX2突变导致CCD患者牙齿替换障碍的病理机制。.本项目收集了CCD患者的原代牙囊细胞，以研究RUNX2突变通过干扰牙囊细胞介导的牙槽骨改建影响牙齿替换的分子机制。结果提示，RUNX2突变通过直接作用下调成骨分化标志基因，负向调控牙囊细胞成骨分化，从而抑制牙齿萌出时的牙槽骨形成，削弱牙齿萌出动力；RUNX2突变还可通过RANK/RANKL/OPG信号通路抑制牙囊细胞诱导破骨细胞分化的能力和抑制单核细胞分化为破骨细胞，导致牙齿萌出过程中骨吸收活性下降，阻碍牙齿萌出通道的建立。这两方面的协同作用导致了CCD患者恒牙萌出障碍。.进一步构建表达野生型或突变型RUNX2的单核细胞，研究RUNX2突变对破骨细胞分化是否有直接调控作用及作用机制。结果提示，RUNX2作为转录因子可以进入细胞核与下游靶基因的DNA 结合，提升mTORC2的表达水平和活性，促进AKT磷酸化，增加NFATc1的表达和入核，从而使NFATc1下游包括CTSK在内的破骨细胞功能相关基因表达增加，最终表现为破骨细胞分化和骨吸收功能的增强。因此，RUNX2通过AKT/NFATc1/CTSK轴促进破骨细胞分化和骨吸收，但是突变型RUNX2发生了功能丧失，导致牙槽骨破骨细胞分化和功能异常，阻碍牙齿萌出通道的建立。.本项目收集到一例不伴有RUNX2突变的CCD患者家系，通过外显子测序初步锁定IGSF10基因变异。随后的功能研究发现，在成骨细胞中敲低IGSF10可显著抑制其矿化潜能，并显著下调成骨相关基因BSP表达水平；恢复IGSF10敲低细胞中BSP表达水平，可挽救成骨细胞的矿化能力。因此，IGSF10可能是一个导致CCD发生的新致病基因，IGSF10可能通过RUNX2之外的通路靶向BSP调控成骨分化，从而导致CCD患者骨代谢异常。

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