牙龈卟啉单胞菌肽酰精氨酸脱亚氨酶与类风湿性关节炎的相关机制研究

The proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) play an important role in development process in RA. Porphyromonas gingivalis (P. gingivalis), a Gramnegative anaerobic black-pigmented rod, is one of the major pathogens associated with periodontitis. P. gingivalis is the only known oral bacterium expressiong the Peptidylarginine deaminase (PAD, coded by PG1424), which leads to the citrullination of rheumatoid arthritis (RA) autoantigen such as fibrin in synovium joint. Moreover, P. gingivalis has also been identified in fibroblast-like synoviocytes (RA FLS) and synovial fluid from patients with RA. Recent findings suggest a causative link between periodontitis and RA via P. gingivalis-dependent induction, but the role of PPAD is controversial. Our previous study found P. gingivalis invaded RA FLS, then increased PG1424 of P. gingivalis and RhoA of RA FLS mRNA expression, promoted the RA FLS cell proliferation, but the regulation mechanism was still unknown. We speculate that PPAD regulate cell biology behavior of RA FLS via RhoA/ROCK signaling pathways, which is a new mechanism of P.g associated with RA. In this study, we will 1) create the PG1424-defective mutant and revertant by allelic exchange, and express and purify P. gingivalis PAD (PPAD); 2) constructe the co-culture model of RA FLS with PG1424-defective mutant or PPAD, then study that PPAD modulate RA FLS’s biological behavior including proliferation, apoptosis, changes of cell cycle, migration, and cellular morpholog via the RhoA/ROCK pathway by inhibiting and activating RhoA/ROCK, and using PCR array, Human Cytokine Antibody Array 1 and bioinformatics technology; 3) constructe RA rat model, analyze that PPAD’s influence on RA rat by using PG1424-defective mutant or revertant infection RA rat model. This study will provide theoretical basis for elaborating the associated mechanism of periodontitis and RA, and raise the new ideas for preventing and treating of periodontitis and RA.

类风湿性关节炎(RA)成纤维样滑膜细胞(FLS)的增殖和迁徙在RA发生发展过程中起重要作用。牙龈卟啉单胞菌(P.g)是牙周病炎重要致病菌，在RA FLS及关节滑液中检出率高。PG1424基因编码肽酰精氨酸脱亚氨酶(PPAD),PPAD的功能是学者研究RA与牙周炎相关关系的焦点,但机制不清存在争议。前期实验发现P.g内化于RA FLS后，PG1424基因上调，细胞RhoA表达上调，促进RA FLS增殖，结果提示PPAD及RhoA参与P.g促进RA FLS增殖过程。我们推测“PPAD通过RhoA/ROCK信号转导通路调控RA FLS增殖、迁徙等细胞生物学行为”是P.g和RA关联的机制之一。本研究拟构建PG1424突变菌株及回复突变菌株，制备细菌与RA FLS共培养模型和细菌感染RA动物模型，应用siRNA、PCR芯片等技术验证假说，为牙周炎和RA的预防和治疗提供新思路。

牙周炎症与类风湿性关节炎的发生发展密切相关。证据充分的牙周致病菌-牙龈卟啉单胞菌（Porphyromonas gingivalis, P. gingivalis）是唯一能够表达肽酰精氨酸脱亚氨酶（peptidylarginine deiminase，PAD）的口腔细菌，在类风湿性关节炎患者纤维样滑膜组织及关节滑液中检出率高。PAD在类风湿性关节炎疾病过程中能够催化精氨酸残基转化为瓜氨酸残基，引发宿主自身免疫反应。P. gingivalis PG1424基因编码肽酰精氨酸脱亚氨酶。近年来国内外学者将P. gingivalis PAD作为牙周炎和RA相关机制研究的焦点，但机制不清存在争议。我们首先构建P. gingivalis PG1424基因突变菌株，对其生物学功能进行初步研究。其次构建类风湿性关节炎和牙周炎双疾病大鼠模型，发现PG1424基因缺失显著下调动物模型血清炎症因子及IL17表达，下调大鼠牙周组织和踝关节炎症因子mRNA水平表达，获得PPAD加重伴类风湿性关节炎牙周炎炎症程度的潜在证据。PG1424基因缺失显著影响动物模型免疫反应，获得PPAD通过Th17/Threg和Th1/Th2途径调控伴类风湿性关节炎牙周炎免疫反应的证据。我们发现PG1424能够下调踝关节滑膜组织凋亡相关基因、RhoA和ROCK mRNA水平表达，获得了PPAD通过RhoA/ ROCK通路上调伴类风湿性关节炎牙周炎踝关节黏膜增殖的潜在证据。我们建立了P. gingivalis PG1424基因突变菌株感染人前髓细胞模型，获得PG1424基因下调炎症因子表达的潜在证据。构建P. gingivalis慢性感染口腔上皮细胞模型，发现核转录因子NF-E2-related factor 2（nrf-2）下调，提示nrf-2可能参与P. gingivalis感染牙周组织。构建P. gingivalis 感染血管内皮细胞模型，探索P. gingivalis 感染对血管内皮细胞炎症表达的调节的潜在证据。通过我们的研究，获得PPAD与RA关联机制的实验证据，希望为牙周治疗利于类风湿性关节炎乃至其他全身疾病的预防及治疗提供有力证据，为牙周炎和RA的预防和治疗提供新思路。