椎间盘退变及纤维化中基质金属蛋白酶12的作用研究

Intervertebral disc (IVD) degeneration is a major cause of low back pain and disability, for which the etiology is not yet clear. Our recent data revealed that fibrosis had a major role in IVD degeneration. Matrix metalloproteinase 12 (MMP12) is a pro-inflammatory and pro-fibrotic factor, as well as a transcription factor. MMP12 could induce the migration of myofibroblasts and macrophages to the injured site. In other diseases, MMP12 depletion retards fibrosis, and has been reported to affect the expression of other fibrosis related MMPs, including MMP2 and MMP3, which are known to be upregulated during IVD degeneration..In a pilot study, we demonstrated that MMP12 was the most significantly upregulated gene in human IVD degeneration among other conventional degeneration indicators. Consistently, we found an increase in the number of cells positive for MMP12 expression in rat and human IVDs during degeneration. These MMP12(+) cells co-expressed α-smooth muscle actin (a-SMA) and fibroblast specific protein 1 (FSP1), which are known to also be expressed by myofibroblasts. More importantly, we found that implantation of mesenchymal stem cells (MSC) could alleviate disc degeneration in part via down-regulating MMP12 expression in degenerated NP cells..We hypothesize that MMP12 has an as yet unidentified impact on disc cell homeostasis through promoting inflammatory signaling and interaction with other MMPs, which results in IVD degeneration and fibrosis. In this proposal, we aim to fully investigate the role of MMP12 in IVD homeostasis. This will be achieved by three objectives: 1) To establish the association of MMP12 positivity with the degree of IVD fibrosis; 2) To elicidate the contribution of MMP12 to disc homeostasis through MMP12 depletion in vitro and in vivo; 3) To investigate the signaling events induced by MMP12 injection..This study will generate novel insights on how MMP12 is involved in the pathogenesis of IVD degeneration. Scientifically, revealing the role of MMP12 in IVD degeneration will help us to unravel the biological events that occur during IVD degeneration and to understand the pathogenesis of IVD degeneration. Clinically, the insights may facilitate future drug development that would benefit millions of patients suffering from low back pain.

椎间盘退变与腰痛有关。我们前期研究发现，椎间盘退变伴随纤维化改变。基质金属蛋白酶12（MMP12）是一个前纤维化因子与转录因子，诱导巨噬细胞和肌成纤维细胞的迁移。在其他系统中，MMP12敲除能减缓纤维化，并影响其他纤维化MMP的表达。我们对椎间盘退变候选基因的筛查表明，退变IVD中MMP12为最显著上调基因，并且MMP12与肌成纤维细胞标记蛋白存在共表达。注射间充质干细胞能降低纤维化并下调MMP12，提示MMP12与椎间盘纤维化存在重要联系。我们推测MMP12可能通过吸引巨噬细胞和促进炎症级联反应导致椎间盘纤维化。本研究将通过：1)确认MMP12与纤维化程度的联系及其细胞来源; 2）MMP12体内外敲除实验; 3）MMP12信号传递研究，来验证MMP12对椎间盘稳态的影响。这是首次关于MMP12与椎间盘退变的系统研究，为退变揭示新信号机制，鉴定新关键致病分子，为阻断退变寻求新手段。

椎间盘退变与腰痛有关。我们前期研究发现，椎间盘退变伴随纤维化改变。基质金属蛋白酶12（MMP12）是一个前纤维化因子与转录因子，诱导巨噬细胞和肌成纤维细胞的迁移。在其他系统中，MMP12敲除能减缓纤维化，并影响其他纤维化MMP的表达。我们对椎间盘退变候选基因的筛查表明，退变IVD中MMP12为最显著上调基因。我们推测MMP12与椎间盘纤维化存在重要联系，MMP12可能通过促进炎症级联反应导致椎间盘纤维化。我们首先使用TGF-beta1进行了髓核细胞的纤维化造模，发现TGF-beta1能刺激髓核细胞表达肌成纤维细胞的表型标记物。然后通过siRNA敲除技术对髓核细胞的MMP12进行基因沉默，发现了MMP12沉默会阻断TGF-beta1的诱导纤维化作用，减少了TGF-beta1诱导导致的髓核细胞的肌成纤维细胞标志物表达。我们进一步进行了MMP12缺陷小鼠的椎间盘诱导退变实验，并与野生型小鼠进行对比，发现在MMP12缺失的情况下，经纤维化穿刺导致的椎间盘纤维化程度明显降低。同时，通过免疫蛋白印迹技术，确认了TGF-β1/Smad轴参与髓核细胞向肌成纤维细胞的转化。另外，我们还发现退变椎间盘中a-SMA表达也发生了升高；分析了炎症因子在疼痛性椎间盘退变进程中的重要作用与相关涉及的信号通路，提出了目前有实验和临床证据支持的有潜力的椎间盘修复候选分子；综合分析了椎间盘内源性祖细胞的分布、性质和特性，以及其受椎间盘退变/衰老与微环境的影响；并汇总了骨代谢疾病中自噬/线粒体自噬的作用。总的来说，本项目支持的研究为椎间盘退变揭示了新信号机制，鉴定新关键致病分子，将有助于为阻断退变寻求新手段。