干预TRAF6对类风湿关节炎成纤维样滑膜细胞增殖、迁移和软骨/骨侵蚀能力的调控作用及机制

Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) play key roles in mediating bone erosion which have erosive ability to destroy cartilage and bone, and could spread the disease to other unaffected joints by hematogenous migration. TNF-α, IL-1β and TLRs plays important roles in mediating the activation, pro-inflammatory cytokine production and matrix destruction of RA-FLS. TRAF6 is the key adapter protein at the downstream of TNF receptor and IL-1/TLRs superfamily signaling pathway. We have found elevated synovial TRAF6 was involved in the pathogenesis of synovitis and bone erosion in RA through modulating synovial inflammation and osteoclast differentiation; TRAF6 expression in RA-FLS was up-regulated, and suppression TRAF6 by siRNA could inhibit the proliferation and pro-inflammatory cytokine production of RA-FLS. However, whether TRAF6 is the 'master switch' mediating the activation and erosive ability of RA-FLS in RA was poorly understood. In this project, we will culture RA-FLS which transfected with TRAF6 plasmids or shRNA by Boyden chamber, set up the David Pretzel in vitro model for evaluating FLS-mediated degradation of intact cartilage, to explore whether TRAF6 regulates the proliferation, immigration, and erosive ability of RA-FLS. Then we will implant RA-FLS which transfected with TRAF6 plasmids or shRNA together with normal human cartilage subcutaneously in severe combined immunode?cient mice (SCID), and inject RA-FLS which transfected with TRAF6 plasmids or shRNA intraarticularly to SCID mice, to further elucidate whether TRAF6 and RA-FLS are involved in the patogenesis of jiont destruction in RA, and to elucidate whether TRAF6 could regulate joint destruction in RA by regulating the proliferation, immigration, and erosive ability of RA-FLS. We expect to find a new therapeutic target for RA and develop new radical drug to inhibit bone destruction more efficiently in RA.

成纤维样滑膜细胞(FLS)是介导类风湿关节炎(RA)关节破坏的关键细胞，最新发现有经血行转移及侵蚀软骨和骨的能力。TRAF6是TNF受体和IL-1/TLR家族下游介导RA-FLS活化和破坏功能的关键分子。我们前一国家自然基金发现TRAF6通过介导炎症反应和破骨细胞分化参与RA滑膜炎症和骨破坏，干扰TRAF6可抑制RA-FLS增殖和产生炎症因子，但TRAF6是否是介导RA-FLS活化和破坏功能的总开关目前尚不明确。本项目拟通过TRAF6质粒或shRNA转染RA-FLS结合Boyden chamber法和David Pretzel体外FLS侵蚀软骨模型法，将转染的RA-FLS±人软骨组织植入SCID小鼠，探讨TRAF6对RA-FLS增殖、迁移和软骨/骨侵蚀能力及对关节软骨/骨破坏的影响，以明确RA-FLS和TRAF6介导的信号通路在RA关节软骨/骨破坏中的作用，旨在寻找新的治疗RA关节破坏靶点

项目的背景：成纤维样滑膜细胞 (FLSs)是介导类风湿关节炎 (RA)关节破坏的关键细胞，最新发现有经血行转移及侵蚀软骨和骨的能力，以FLSs为靶点正成为目前研究RA关节破坏的前沿。TNF-α、IL-1β和TLRs在RA-FLS活化以及促炎和基质破坏功能中起着重要作用。TRAF6是TNF受体和IL-1/TLR家族下游介导RA-FLS活化和破坏功能的总开关。我们在2010年国家自然科学基金青年基金项目(项目编号：81001334)资助下，发现TRAF6可通过介导炎症反应和破骨细胞的分化参与RA滑膜炎症和关节破坏。那么，TRAF6介导的信号通路是否对RA-FLSs增殖、迁移和侵蚀软骨/骨的能力具有调控作用？抑制TRAF6能否通过调控RA-FLSs抑制RA关节破坏？目前尚未见相关报道。.主要研究内容：本研究探讨了TRAF6在RA-FLS中的表达及对其致炎作用的影响，并进一步探讨了TRAF6介导的信号通路对RA-FLSs的软骨/骨侵蚀能力的调控作用。.重要结果和关键数据：我们研究发现，TRAF6可明显表达于滑膜衬里层和衬里下层CD55+ FLSs和其它CD55- 细胞中。原代培养的RA-FLSs中TRAF6表达明显强于OA-FLSs。抑制TRAF6可降低RA-FLSs中炎症因子TNF-α、IL-1β、IL-6和IL-8，以及MMP3和MMP13 mRNA的表达，并降低细胞培养上清液中这些蛋白的水平，也可明显抑制RA-FLSs增殖，还可将RA-FLSs阻滞在G1期，阻止细胞进入S期，从而抑制RA-FLSs细胞增殖。并且，抑制TRAF6后RA-FLSs细胞迁移的速度明显低于未抑制组。进一步研究显示：TRAF6介导的信号通路参与了RA-FLSs对软骨/骨侵蚀能力的调控作用，阻断TRAF6可减轻RA-FLSs对软骨/骨的侵蚀。我们还发现，RA患者存在骨吸收增加、骨代谢失衡。RA患者滑膜TRAF6表达增加可能与RA代偿性骨形成增加有关，TRAF6可能通过介导滑膜炎症参与了RA的骨代谢失衡。.科学意义：这些研究结果进一步提示抑制TRAF6可能是治疗RA的新的靶点。为RA的治疗提供了新的思路，为RA患者关节软骨/骨破坏的治疗提供了一定的实验理论依据。