Runx2在颞下颌关节骨关节炎软骨细胞分化和基质降解中的作用研究

Osteoarthritis (OA) of temporomandibular joint (TMJ) is the severe form of temporomandibular joint disorders. It is characterized by accelerated degradation of extracellular matrix and differentiation of chondrocytes. Studies show that Runx2 plays key regulation roles on OA development. Whether intervening Runx2 expression in chondrocytes alleviates TMJ OA is unclear. This project will develop abnormal dental occlusion in Tamoxifen induced cartilage-specific Runx2 KO mice and observe the changes of cartilage phenotype, regulation of chondrocytes differentiation and matrix degradation compared with those in wildtype mice. In addition, after development of unilateral anterior crossbite (UAC) on wildtype mice, local injection of AAV5 (Adeno Associated Virus 5)-Runx2 into TMJs will be employed to demonstrate if TMJ OA induced by abnormal dental occlusion will be exacerbated by Runx2 overexpression. Moreover, chondrocytes isolated from TMJ condylar cartilage of mice and ATDC5 cells (chondrocyte cell line) will be treated with Runx2 shRNA (knockdown Runx2) or lentiviral transfected with Runx2 (overexpression of Runx2) and then mechanical loading will be applied on these chondrocytes to confirm the molecular mechanism in activation of Runx2 signaling pathway. This project aims to clarify the regulative role of Runx2 in abnormal chondrocyte differentiation and matrix degradation and it may provide a new targeted approach for OA.

颞下颌关节（TMJ）骨关节炎（OA）是颞下颌关节病的重症表现，其主要病理特征为软骨细胞过度分化和基质过度降解。初步研究表明Runx2在OA发生发展中起重要作用，干预软骨细胞中Runx2的表达是否可以减轻TMJOA尚不明确。本项目拟采用诱导型Runx2软骨特异性敲除小鼠，建立异常咬合所致的TMJOA模型，通过与野生鼠对比，探讨Runx2软骨特异性敲除对TMJ软骨退变的影响；建立野生型小鼠TMJOA模型，通过TMJ关节腔注射腺病毒转染的Runx2，明确上调Runx2表达是否会加重TMJOA；采用离体培养髁突软骨细胞，通过基因转染过表达或下调Runx2，再施以力刺激，明确Runx2在软骨细胞中的活化特点，最终从基因、细胞、动物疾病模型、机械应力多水平阐述Runx2在TMJOA发病中的作用，为TMJOA靶向治疗提供实验依据。

在软骨发育和胚胎期下颌髁状软骨发育过程中，Runx2对于软骨细胞的发育和成熟至关重要。为了明确Runx2是否调节出生后髁突软骨以及椎间盘的生长和组织稳态，我们敲除了出生后小鼠软骨细胞中的Runx2基因，并评估了该敲除对颞下颌关节软骨以及椎间盘生长和改建的影响。组织学和免疫组织化学数据显示Runx2缺乏可引起髁突软骨组织紊乱，包括肥大软骨细胞丢失和肥大区减少、增殖软骨细胞减少以及软骨基质生成减少。在Runx2敲除小鼠中Col10a1、Mmp13、Col2a1、Aggrecan和Ihh的表达明显降低。这项研究的结果表明Runx2是颞下颌关节髁突软骨中软骨细胞增殖和肥大以及出生后颞下颌关节软骨生长和体内稳态所必需基因，Runx2在调节软骨细胞来源的软骨下骨改建中起重要作用。.同时，我们还发现在两周大的小鼠软骨细胞中敲除Runx2基因，在小鼠3个月大时，其髓核组织中有大量幼稚的泡沫状的脊索细胞（notochordral cells)；而在Cre-对照小鼠的髓核组织中则是大量的软骨细胞样细胞（chondrocyte-like cells）；同时敲除鼠的椎间盘软骨组织也比正常对照组更厚；除此之外，我们还发现敲除鼠的髓核细胞核纤维环细胞中的Ihh的蛋白表达也明显增强。这些结果证明，Runx2在出生后小鼠的髓核细胞从幼稚的脊索细胞向软骨细胞样细胞转变过程中必不可少，它在椎间盘组织的发育和稳态中发挥重要作用，并且此作用可能与Ihh信号通路相关。.另外，本课题组还针对骨关节炎（OA）的早期病理变化及发生机制进行了长程观察和分析。我们的数据表明，滑膜炎与OA相关，并且是OA发生和发展的领先事件，因此滑膜可能是OA治疗的重要靶向组织。