肌球蛋白轻链3通过Notch信号在维持软骨稳态中的作用及其机制研究

The senescence and degradation of articular cartilage are important pathological features of osteoarthritis (OA), but the mechanism of its occurrence has not been fully elucidated. In our previous studies, proteomic techniques were used to screen the changing proteins related to cartilage senescence and degeneration. We noticed that the expression of myosin light chain 3 (MYL3) in articular cartilage of aged mice was significantly decreased. Interfered the MYL3 in chondrocytes cell lines, the expression of cartilage degrading enzymes such as MMP13 was significantly increased, while the expression of metabolic factors such as SOX9 was significantly decreased. RNA-sequencing results reminded that MYL3 was associated with Notch signaling. These data suggested that MYL3 may be involved in regulating the synthesis and catabolism of articular cartilage, senescence and degeneration, and even the occurrence of OA through Notch signaling. In order to clarify the role of MYL3-Notch signaling in maintaining the cartilage homeostasis and OA, this project plans to construct a mouse and cell model with inducible knock out MYL3 in chondrocyte. Using RNA sequencing and proteomics and other cell and molecular biological techniques to explore the specific mechanism of MYL3-Notch signal in cartilage senescence and OA at the global, cellular and molecular levels. The aim of this project is to provide a new perspective for cartilage biology and a new potential target for the prevention and treatment of OA.

关节软骨的衰老退变与损伤降解是骨关节炎(OA)的重要病理特征，但其发生的机制尚未完全阐明。课题组在前期研究中利用蛋白质组技术筛选与软骨衰老退变有关的蛋白质，发现老年小鼠关节软骨肌球蛋白轻链3(MYL3)表达显著降低，干扰软骨细胞中MYL3后软骨降解酶类MMP13等表达显著增加，软骨合成代谢因子SOX9等表达显著下降，进行RNA测序发现MYL3与Notch信号存在一定联系，提示MYL3可能通过Notch信号参与调节关节软骨的合成及分解代谢，衰老退变乃至OA的发生。本项目拟构建可诱导、软骨细胞中敲除MYL3的小鼠与细胞模型，以明确MYL3-Notch信号在维持软骨稳态、关节软骨衰老退变与OA发生中的作用，应用RNA测序与蛋白质组学等细胞与分子生物学技术，从整体、细胞与分子水平探讨MYL3-Notch信号在软骨衰老与OA发生的中具体机制，旨在为软骨生物学提供新观点，为防治OA提供新的潜在靶点。

骨关节炎（Osteoarthritis，OA）是目前世界上最常见的关节疾病，其病变累及整个关节结构，以关节软骨的损害为最主要特征，是老年人群致残的主要原因。目前我国OA患者已达1.5亿人，随着人口老龄化进程的加速患者数量将进一步增加。由于OA的发病机制尚未阐明，目前尚无有效治疗药物，仅通过综合治疗措施改善症状、减轻疼痛，严重影响患者的生活质量，带来巨大的经济和社会负担。因此，开展针对衰老因素对软骨细胞在OA病程中的作用及其相关信号通路的进一步研究，阐明OA的发病机制，探寻早期的诊断及寻找治疗靶点，对我国众多的OA患者，尤其是老年人群中潜在的OA患者，具有十分重大的意义。在本项目的支持下，取得了以下研究发现：利用蛋白质组学筛查发现并验证了肌球蛋白轻链3（MYL3）在衰老的软骨细胞中MYL3表达下降，同时伴随CME的表达增强，确定了在软骨细胞中MYL3及Clathrin调节内吞（CME）与衰老和OA存在一定联系；细胞及动物实验发现了在MYL3可以通过抑制CME来抵抗衰老，构建软骨细胞特异性敲除MYL3的小鼠并且结合DMM-OA模型，发现MYL3缺失加速软骨细胞衰老和OA进展；机制探索发现MYL3的缺失促进CME介导的Notch信号内吞，激活Notch信号并且诱导软骨细胞发生衰老，加速OA软骨基质降解；使用Notch信号抑制剂RO4929097处理可以改善MYL3缺失导致的OA加重。我们的研究首次发现了软骨细胞中MYL3与Notch-CME-衰老之间的联系，初步阐明MYL3在维持软骨稳态、关节软骨衰老中的作用和机制，为软骨生物学提供了新的观点；确定了MYL3-Nocth信号在OA中的重要作用，发现靶向增强MYL3表达及药物抑制Notch-CME信号可以有效改善软骨细胞衰老及OA发展，MYL3-Nocth为治疗OA的潜在新靶点，为临床治疗OA提供新的思路和办法。