ADAMTS-5在腰椎软骨终板退变中的作用和机制研究

结题报告

项目介绍

AI项目解读

基本信息

批准号：
81271971
项目类别：
面上项目
资助金额：
68.0万
负责人：
范顺武
依托单位：
浙江大学
学科分类：
H0604.骨、关节、软组织损伤与修复
结题年份：
2016
批准年份：
2012
项目状态：
已结题
起止时间：
2013-01-01 至2016-12-31

项目参与者：
周志杰；赵兴；王吉莹；张剑；胡子昂；陈喜君；蔡宏歆；何勇；
关键词：
退变解聚素及金属蛋白酶伴血小板反应蛋白模序-5 腰椎软骨终板

项目摘要

Lumbar cartilage endplate degeneration is closely associated with the process of the lumbar intervetebral disc degeneration and may contribute to low back pain. However, its mechanism remains unclear. ADAMTS-5 is the primary aggrecanase responsible for aggrecan degradation in animal models of osteoarthritis, which also plays a key role in human articular cartilage destruction. Yet its role in human lumbar cartilage endplate remains largely unknown. With our efforts on understanding the molecular mechanisms of lumbar cartilage endplate degeneration, we identified upregulation of ADAMTS-5 in degenerated endplate samples. We further successfully generated an lumbar cartilage endplate degeneration animal model by intradiscal injection of IL-1β. And the injection of IL-1β enhanced ADAMTS-5 expression and subsequently caused extracellular matrix degeneration. This observation was further confirmed by our ex vivo data that demonstrated IL-1β induced endplate degeneration via upregulation of ADAMTS-5. Previous study revealed that IL-1β-NF-kB signalling pathway could induce articular cartilage degeneration via upregulation of ADAMTS-5. Based on these findings, we propose to investigate the effect of ADAMTS-5 on lumbar cartilage endplate degeneration using a chondrocyte specific ADAMTS-5 conditional knockout mice, as well as lentivirus mediated ADAMTS-5 gene overexpression. We believe we will have interesting histologic, cytologic and molecuar findings.In addition, we aim to investigate the regulatory mechanism of ADAMTS-5, which may be regulated by IL-1β through the NF-kB signalling pathway. Our study may probably provide a theoretical foundation for targeted biological therapy for the treatment of lumbar cartilage endplate degeneration.

腰椎软骨终板退变与腰椎间盘退变及下腰痛密切相关，其发病机制仍不明确。ADAMTS-5是骨关节炎动物模型中最重要的关节软骨细胞外基质蛋白降解酶，也是人关节软骨退变过程中关键的基质蛋白酶，但在腰椎软骨终板退变中的作用未见报道。我们前期研究发现患者退变腰椎软骨终板中ADAMTS-5的表达显著升高，进一步动物模型和细胞学研究表明IL-1β可使ADAMTS-5的表达增加，降解细胞外基质，导致终板退变，提示ADAMTS-5在腰椎软骨终板退变中发挥重要作用。文献表明关节软骨中ADAMTS-5的表达可被IL-1β-NF-kB通路信号调控，并导致关节软骨退变。为此，本研究将以ADAMTS-5条件性基因敲除和基因过表达为基础，从组织、细胞及分子层面分析ADAMTS-5在腰椎终板退变中的作用。并通过NF-kB通路研究IL-1β调节ADAMTS-5的可能机制，为开发以ADAMTS-5为靶点的生物治疗提供理论依据。

结项摘要

腰椎软骨终板是椎间盘的营养通道门户，其退变与腰椎间盘退变及下腰痛密切相关，其发病机制仍不明确。我们在课题申报时提出关节软骨细胞外基质蛋白降解酶ADAMTS-5在腰椎软骨终板退变中也起着重要的作用，且其表达可能受到炎性因子IL-1β及其下游NF-kB 信号通路调节。我们收集了60例人腰椎软骨终板标本，通过组织学染色和RT-PCR验证了带有Modic改变的退变软骨终板和正常软骨终板基质表达和组织学差异，通过RT-PCR和免疫组化检测ADAMTS-5、ADAMTS-4和IL-1β表达，发现退变组织中ADAMTS-5表达显著升高，ADAMTS-4表达未见明显改变，IL-1β的表达明显升高。在腰椎软骨终板细胞中，我们建立了ADAMTS-5过表达细胞模型，通过RT-PCR和Western blot检测终板软骨细胞合成代谢和分解代谢的改变，发现分解代谢明显上调，而合成代谢基本下调，TIMPs也显著上调。我们在体外培养的大鼠腰椎间盘模型中，应用IL-1β促进其退变，以2μM和10μM的ADAMTS-5抑制剂114810同时应用，发现抑制ADAMTS-5的活性可以保护腰椎软骨终板。在腰椎软骨终板细胞中，我们通过Western blot、RT-PCR和免疫细胞化学均发现IL-1β刺激可以浓度依赖和时间依赖性地促进ADAMTS-5的表达，而对ADAMTS-4作用不明显。而应用NF-kB 信号通路抑制剂SN50可减弱IL-1β对ADAMTS-5的刺激作用。Western blot 实验验证IL-1β可以激活NF-kB 信号通路。染色质免疫共沉淀（ChIP）进一步验证NF-kB与ADAMTS-5启动子的相应区域结合。该课题研究结果证明了ADAMTS-5在腰椎软骨终板退变中的重要作用且IL-1β可以通过NF-kB信号通路调节ADAMTS-5表达。ADAMTS-5及其上游信号通路可作为治疗腰椎软骨终板退变的靶点，具有转化应用价值。