纤维结构不良中破骨细胞过度激活的分子机制研究

Fibrous dysplasia (FD), caused by GNAS mutation, is one of common diseases in orthopaedic clinic, which is characterized by the replacement of normal bone with abnormal fibro-osseous tissue. Excessive activation of osteoclasts is observed in FD lesion, which is believed to be one of the most important histological manifestations of FD absolutely. The results from our preliminary research showed that osteoclasts differentiation from mononuclear macrophage was significantly improved by GNAS mutation (R201H), in which the up-regulation of IL6 and RANKL in MSCs and the activation of Ca2+-calmodulin-calcineurin and RANK-TRAF6-TAB2/TAK1 signaling pathways in mononuclear macrophage were involved. Therefore, this project is designed to elucidate the molecular mechanisms underlying how MSCs with GNAS mutation could improve osteoclasts differentiation through the up-regulation of IL6 and RANKL in osteoblasts-osteoclasts interaction mode and why osteoclasts differentiation is improved in response to GNAS mutation in mononuclear macrophage through the activation of Ca2+-calmodulin-calcineurin and RANK-TRAF6-TAB2/TAK1 signaling pathways. Transgenic mouse model with MSCs- and osteoclasts-specific conditional GNAS mutation (R201H) knock-in will be constructed, by which we make an attempt to confirm the in vitro data and rescue the FD phenotype. This study provides useful insight into the signaling pathways involved in the FD phenotype and facilitates dissection of the molecular pathogenesis of FD and testing of novel therapies.

纤维结构不良（FD）病因为GNAS突变，特征为编织骨由纤维组织化生形成，破骨细胞过度激活。前期研究发现，GNAS突变上调MSCs中IL6和RANKL表达和激活单核巨噬细胞Ca2+-calmodulin-calcineurin和RANK-TRAF6-TAB2/TAK1通路而促进破骨细胞分化。因此本项目以“FD破骨细胞过度激活”为线索，以MSCs和单核巨噬细胞中GNAS突变为切入点，从成骨细胞-破骨细胞相互作用和破骨细胞本身两个角度研究“MSCs中GNAS突变如何上调IL6和RANKL的表达从而促进破骨细胞分化”和“单核巨噬细胞中GNAS突变如何通过Ca2+-calmodulin-calcineurin和RANK-TRAF6-TAB2/TAK1通路促进破骨细胞分化”等问题，探索FD中破骨细胞过度激活的机制。并构建破骨细胞和MSCs特异的GNAS突变敲入小鼠对结果进行验证并对FD表型进行挽救。

纤维结构不良病因为GNAS突变，特征为编织骨由纤维组织化生形成，破骨细胞过度激活。本项目以“FD破骨细胞过度激活”为线索，以MSCs和单核巨噬细胞中GNAS突变为切入点，从成骨细胞-破骨细胞相互作用和破骨细胞本身两个角度研究“MSCs中GNAS突变如何上调IL6和RANKL的表达从而促进破骨细胞分化”和“单核巨噬细胞中GNAS突变如何通过Ca2+-calmodulin-calcineurin和RANK-TRAF6-TAB2/TAK1通路促进破骨细胞分化”等问题，探索FD中破骨细胞过度激活的机制。研究结果证明：GNAS基因突变的MSCs可显著地促进破骨细胞分化；IL6和RANKL是LV-R201H hMSCs促进破骨细胞分化的关键靶点；GNAS基因突变的单核巨噬细胞向破骨细胞分化的能力显著增强；Ca2+-CalM-CN通路和RANK-TRAF6-TAB2/TAK1通路是介导突变GNAS增强RAW264.7破骨分化能力的关键靶点。而且，围绕纤维结构不良中出现的“间充质干细胞成骨分化障碍”，本项目亦进行了更深层次的研究。从表观遗传修饰和能量代谢两个角度，我们发现：间充质干细胞成骨分化过程中成骨标志基因的启动子区发生动态的和特异的表观遗传学修饰，这些修饰与成骨标志基因的表达谱紧密相关；糖皮质激素骨质疏松小鼠的骨髓间充质干细胞PPARγ基因启动子区存在特异的表观遗传修饰，这些修饰与骨质疏松干细胞成脂分化潜能增强有关；AMPK-Gfi1-OPN信号轴介导了AMPK对干细胞成骨-成脂分化平衡的调节。本项目的研究深化了人们对纤维结构不良病理生理学表型的认识，揭示了间充质干细胞定向分化的分子机制。