模式识别受体在牙周组织和细胞的炎症反应及组织破坏机制中的作用和信号通路机制的研究

Immunal response initiated by periodontal pathogens is one of the pathogenesis of periodontitis. Pattern recognition receptors (PRRs) can recognize pathogen associated molecular patterns (PAMPs), which are important for triggering innate immune response in many inflammatory dieases. However, the role of PRRs except Toll-like receptors in periodontitis is not clear. In our previous study, it was found that besides Toll-like receptors, the level of other PRRs (such as NOD-like receptors) as well as adhesion molecules and RANKL were increased in periodontal fibroblasts when stimulated by Porphyromonas gingivalis. Therfore we hypothesize that PRR may play an important role in pathogenesis of periodontitis. In this study, the whole PRR gene expression profile in gingival tissue from periodontitis patients and in periodontal fibroblasts treated by P.gingivalis will be analyzed by PT2 real-time PCR arrays. The PRRs which may take part in periodontitis pathogenesis will be selected and to be testified further by real-time PCR and Western-blot techniques. Then the roles of these PRRs and signal pathway mechanisms in expression of inflammatory cytokines (such as adhesion molecules), tissue-break-down media (such as matrix metalloproteinases and RANKL) will be studied by applying special receptor agonists and knocking down these PRRs gene. The results of this study will supply more evidences for our understanding the role of PRR in periodontitis, and enrich the theory of periodontal pathogenesis. Besides, it could provide clues and set up theoretical basis for a new treatment strategy of periodontal diseases.

牙周致病菌引发的免疫炎症反应是牙周炎的致病机制之一，模式识别受体（PRR）是机体识别病原体相关分子模式从而启动固有免疫应答的重要途径。我们前期探索用牙龈卟啉单胞菌（Pg）刺激牙周组织细胞，发现除Toll样受体外还有多种PRR高表达，粘附分子、RANKL等破坏因子也高表达，推测PRR在牙周炎致病机制中会发挥重要作用。本课题拟通过实时荧光定量PCR芯片技术观察PRR在牙周炎组织及Pg感染的细胞中的表达全貌；利用实时定量PCR、蛋白免疫印迹等技术验证筛出的PRR在牙周炎组织和感染细胞中的表达；并利用特异的PRR激动剂和基因沉默技术从正反两方面探讨相关PRR在Pg促牙周组织细胞表达粘附分子等炎症因子和基质金属蛋白酶、RANKL等组织破坏介质中的作用及可能的信号通路，全面、深入地阐述PRR在牙周炎症反应和软硬组织破坏中的作用及机制，丰富牙周炎致病机制的理论体系，为建立新的治疗策略提供线索和理论基础。

牙周致病菌引发的免疫炎症反应是牙周炎的致病机制之一,模式识别受体（PRR）是机体识别病原菌进而启动固有免疫应答的重要途径，但在牙周炎致病机制中仅对Toll样受体的作用有所认识,其他PRR的作用并不清楚。本项目收集了慢性牙周炎和侵袭性牙周炎患者及各自健康对照者的牙龈组织，并用牙龈卟啉单胞菌（Pg）刺激人牙周膜成纤维细胞（hPDLCs）及人牙龈成纤维（hGFs），采用高通量方法检测了上述组织和细胞中PRR的表达；对筛选出的NOD1和NOD2进行了深入研究。通过免疫组化、Real time-PCR、特异PRR激动剂和基因沉默技术、特异信号通路抑制剂、流式细胞术和蛋白印迹等方法，观察了牙龈组织中NOD1和NOD2的表达分布特点；检测了NOD1和NOD2在hPDLCs和hGFs受到Pg刺激后表达炎症因子、粘附分子、MMP和TIMPs、RANKL/OPG中发挥的作用及信号通路；并研究了Toll 4、NOD1和NOD2在单核细胞系U937细胞与hPDLCs和hGFs粘附中的功能和机制、及硫化氢在hPDLCs和hGFs表达IL-6、IL-8中的作用。主要结果：1）牙周炎的牙龈组织中大多数Toll样受体和NOD样受体的表达高于健康对照；2）NOD1和NOD2在慢性牙周炎的牙龈结缔组织中表达增高，强阳性区集中在浸润的炎症细胞及周围的成纤维细胞中；3）NOD1和NOD2在hPDLCs和hGFs受到Pg感染后表达增高，证实其通过NF-κB及MAPK信号通路在这两种细胞表达IL-6、IL-8和ICAM-1、VCAM-1中发挥重要作用。4）NOD1和NOD2能诱导U937细胞表达粘附分子FLA-1和VLA-4，介导了单核细胞与hPDLCs和hGFs的粘附；5）牙周炎牙龈组织中NOD2的表达与RIPK2的表达显著正相关。6）Pg感染hPDLCs和hGFs可调控多种MMP、TIMPs及RANKL/OPG的表达，但NOD1和NOD2在其中未发挥作用。7）硫化氢在hGFs和hPDLCs表达IL-6、IL-8中发挥了作用，通路是NF-κB信号通路。这些结果揭示了NOD1和NOD2在牙周炎致病机制中的作用及信号通路机制，首次证明了NOD1和NOD2与粘附分子之间的关系，扩展了对PRR功能的认识，丰富了牙周炎致病机制的理论体系，为今后发展新的防治策略提供了理论依据。