FGFR3负性调控软骨向成骨细胞转分化影响骨性关节炎软骨下骨硬化的作用与机制研究

Articular cartilage degeneration and subchondral sclerosis are the critical pathogenesis contributes to osteoarthritis (OA) progression. Our previous studies reveal that fibroblast growth factor receptor 3 (FGFR3) plays a chondroprotective role in joints. However, the mechanisms of FGFR3 in chondrocytes on the influence of subchondral sclerosis during OA progression is still unknown. Recently, it has been found that chondrocyte-to-osteoblast transformation during endochondral ossification is regulated by the induction of pluripotency factors, including Oct4 and Sox2, in chondro-osseous border. The results of our preliminary studies showing the specific expression of Oct4 and Sox2 is detected in OA calcified cartilage zone, and which is negatively regulated by FGFR3 in chondrocytes. Additionally, inhibition of PI3K/AKT signaling ameliorates cartilage defects and downregulates the expression of Oct4 in calcified cartilage zone of FGFR3-deficient mice. Presumably, downregulated expression of chondrocytes FGFR3 in OA upregulates PI3K/AKT signaling which induce the expression of pluripotency factors, and promotes chondrocyte-to-osteoblast transformation, and upregulates ossification progression which led to subchondral sclerosis. To confirm this hypothesis, we are planning to investigate the effects and mechanisms of FGFR3 on the regulation of subchondral bone remodeling during OA progression from in vivo and in vitro, which will provide the experimental data for developing FGFs/FGFR3 signaling based biological treatment to prevent or attenuate OA.

软骨退变与软骨下骨硬化是骨性关节炎（OA）发病的关键环节。我们前期发现：FGFR3在OA中发挥软骨保护作用，而软骨细胞内FGFR3负性调控软骨下骨重建影响软骨下骨硬化的机制不详。近期文献提示：软骨细胞可转分化为成骨细胞，且在转分化区特异性表达的多能分子—Oct4和Sox2促进这一过程。我们发现：FGFR3负性调控OA关节矿化软骨区特异性表达的Oct4和Sox2，且抑制PI3K/AKT信号活性可缓解FGFR3敲除所导致的矿化软骨区病变及Oct4高表达。据此推测：OA软骨细胞内FGFR3降低可能通过上调PI3K/AKT信号促进多能分子表达，促进软骨向成骨细胞转分化，加快软骨下骨形成，进而加重软骨下骨硬化。本课题拟利用多种基因修饰动物模型，通过表型分析以及细胞谱系示踪实验，结合细胞水平实验，阐明FGFR3影响OA软骨下骨硬化的作用与机制，为从干预FGFs/FGFR3信号角度防治OA提供实验依据。

背景:Vismodegib作为一种外源性的印度hedgehog拮抗剂，已被FDA批准用于基底细胞癌患者的临床治疗，此前的观察表明Vismodegib在骨关节炎治疗中的潜在疗效。然而，还没有直接证据表明Ihh在成年小鼠的椎间盘稳态中发挥作用。本研究的目的是评估成年期全身给予Smoothened inhibitor (SMOi) - Vismodegib对椎间盘稳态的影响。..方法:采用免疫组化方法检测SMOi全身给药后成年小鼠ivd中Ihh信号转导靶基因Gli1的表达。通过x线和micro-CT观察SMOi治疗后椎体的病理变化。通过组织学分析评价SMOi对包括软骨终板、生长板和纤维环在内的ivd内稳态的影响。免疫组化检测Aggrecan、MMP13和Runx2在ivd中的表达。通过TUNEL检测和Ki-67表达分析来评估ivd中软骨细胞凋亡和增殖的变化。..结果:全身施予SMOi可显著降低ivd中Gli1的表达，表明SMOi可有效抑制Ihh信号通路。SMOi治疗后椎体骨量减少。此外，在smoi治疗小鼠的ivd中，可以观察到idd样缺陷，包括椎体骨量减少、CEP硬化和退行性NP，以及房颤内GP和裂隙变窄或融合。IDD的严重程度与2-8周后给予SMOi治疗的时间有关。SMOi治疗小鼠ivd中Aggrecan、MMP13和Runx2的表达均显著降低。此外，软骨细胞凋亡明显增强，软骨细胞增殖明显受到抑制。..结论:本研究提示Ihh信号通路是成年期维持ivd软骨结构完整性和功能所必需的重要信号通路。