PTH1R/Zfp467信号轴调控颌骨骨髓间充质干细胞成骨分化影响牙萌出动力的机制研究

Heterozygous mutations in the parathyroid hormone 1 receptor (PTH1R) gene were shown to be associated with primary failure of tooth eruption. The tooth fails to erupt to the occlusal plane due to lack of motive force. Alveolar bone formation at the base of the tooth is required for eruption by providing motive force, whereas the underlying mechanism was not well-understood. We have previously generated orofacial bone marrow stem cells (OMSC) specific deletion of PTH1R mouse model. The mutant mice exhibited disturbance of tooth eruption, accompanied by significantly reduced alveolar bone formation at the base of the tooth when compared to control littermates. The expression of a novel PTH1R signaling downstream factor, Zinc finger protein 467 (Zfp467) was statistically reduced during tooth eruption. PTH directly down-regulated Zfp467 while Zfp467 expression was increased in alveolar bone of mutant mice devoid of PTH1R expression. The potential role of PTH1R/Zfp467 signaling in OMSC during tooth eruption has yet not been investigated. To address these questions, the current study aims at understanding the mechanism of Zfp467 in regulating osteogenesis of OMSC using RNA interference and ChIP-Seq techniques. Zfp467 flox mice will be generated via CRISPR/Cas9-mediated genome editing technology to further elucidate the in vivo function of PTH1R/Zfp467 signaling in bone remodeling during tooth eruption. The results will provide a potential treatment target to control bone formation in patients with disturbances of tooth eruption.

人体甲状旁腺激素1受体（PTH1R）基因功能缺失引起原发性牙萌出障碍，患牙表现为萌出动力不足。牙基底部骨形成是萌出动力的主要来源之一，但其调控机制尚不清楚。申请人构建了颌骨骨髓间充质干细胞（OMSC）特异性PTH1R敲除鼠，发现突变鼠牙萌出障碍，牙基底部骨形成下降导致萌出动力不足。锌指蛋白467（Zfp467）是PTH1R通路下游因子，但在OMSC与牙萌出中的作用鲜有报道。申请人发现Zfp467受PTH负调控，随牙萌出及成骨分化表达下调，PTH1R敲除导致牙槽骨Zfp467表达增加。本项目拟根据前期结果，通过慢病毒介导的Zfp467表达改变分析其在OMSC成骨分化中的作用，通过ChIP-Seq技术筛选Zfp467下游靶基因，利用CRISPR/Cas9构建Zfp467条件敲除鼠阐明PTH1R/Zfp467信号轴调控牙萌出动力的机制，为探索通过重建萌出动力治疗牙萌出异常的新干预靶点提供线索。

牙萌出是指牙冠形成后向合平面移动，穿过牙槽骨和口腔黏膜到达功能位置的一系列复杂生理过程。目前认为牙萌出的机制主要涉及牙萌出通道的形成与牙萌出动力。干细胞介导的牙槽骨骨改建在建立萌出通道以及提供萌出动力中发挥重要作用。研究表明甲状旁腺激素受体（Parathyroid hormone 1 receptor, PTH1R）基因功能缺失导致原发性牙萌出障碍，但其作用机制尚未完全明确。本课题利用谱系示踪实验明确了颌骨骨髓间充质干细胞在颌面部的表达谱及分化命运；通过转基因小鼠技术构建了颌骨骨髓间充质干细胞特异性基因敲除小鼠，通过micro-CT、骨组织形态计量学实验、免疫组化染色及共聚焦显微镜技术等分析牙萌出表型，探索PTH1R/Zfp467信号轴对牙萌出过程中牙槽骨骨形成的体内调控作用；对颌骨骨髓间充质干细胞的分化、增殖等细胞学特性进行全面系统的探究；通过高通量测序技术、细胞学技术、RNA干扰技术深入探索了PTH1R/Zfp467对颌骨骨髓间充质干细胞成骨向分化的影响及其分子机制。本项目的开展不仅有助于阐明牙萌出的分子生物学机制，也有望为牙萌出异常的早期诊断与临床治疗提供新思路。