RA患者滑膜液ACPA-IC介导成纤维样滑膜细胞增殖及细胞因子释放的机制研究

The presence of immune complexes (IC) and anti-citrullinated protein antibodies (ACPA) are abundant in serum and synovial fluid (SF) of RA patients, which is likely to contribute to the pathogenesis of RA and to articular damage. Since they are able to activate the complement, stimulate phagocytes through their Fc receptor and release cytokines (CKs), metalloproteinases (MMPs) and reactive oxygen intermediates. Based on the previous study findings that ACPA-IC from RA sera could stimulate the proliferation of fibroblast-like synoviocytes (FLS) and promote the release of inflammatory cytokines, we would continue to investigate the expression of ACPA-IC on synovial tissues from patients with RA by immunohistochemistry; extract the ACPA-IC from synovial fluid (sfACPA-IC) using affinity chromatography and identify the antigens of sfACPA-IC utilized Western blot or ELISA; screen and identify the citrullinated antigens in sfACPA-IC applied the MALDI-TOF-MS; study the effects of FLS proliferation and CKs and MMPs secretions stimulated by FLS cultured in vitro; analyze the activation of signaling pathway components (NF-κB, p38, JNK, ERK. etc) which mediated the FLS proliferation and inflammatory cytokines secretion. The above work would further explore the mechanism and roles of sfACPA-IC in RA synovitis, and we hope to provide breakthrough diagnostic indicators and therapeutic targets for RA.

RA患者血液及关节滑膜液（SF）中存在大量免疫复合物（IC）及抗瓜氨酸化蛋白抗体（ACPA），IC通过激活补体、活化吞噬细胞并释放细胞因子（CK）等炎性介质，引起滑膜炎性损伤。本课题拟在前期研究血清ACPA特异性IC（ACPA-IC）刺激成纤维样滑膜细胞（FLS）增殖及CK释放的基础上，研究ACPA-IC在RA滑膜组织中的表达；从RA SF中提取ACPA-IC（sfACPA-IC），MALDI-TOF-MS等法鉴定sfACPA-IC中瓜氨酸化抗原成分；体外培养FLS，研究sfACPA-IC对FLS增殖的影响；液态芯片法分析sfACPA-IC对FLS分泌CK和基质金属蛋白酶(MMP)的影响；研究sfACPA-IC介导FLS增殖及炎性因子分泌的信号通路分子的变化。上述研究可望进一步阐述sfACPA-IC在RA滑膜炎性损伤中的作用及其机制，为探讨RA新的诊断和治疗分子靶标提供理论和实验依据。

RA患者血液及滑膜组织/液（ST/SF），中存在的ACPA及其免疫复合物（IC）在滑膜组织炎性损伤中发挥重要作用。本课题在前期研究RA血清ACPA-IC对成纤维样滑膜细胞（FLS）功能影响的基础上，进一步研究ACPA-IC在RA滑膜组织中的表达情况；用LC-MALDI-TOF/TOF、MALDI-TOF-MS、HPLC-Thermo-fisher LTQ-Orbitrap等系列高精度质谱技术筛选鉴定RA血清、ST及SF的IC（S-IC、ST-IC、SF-IC）中的差异蛋白及瓜氨酸蛋白（CitP）成分。取得了以下成果：（1）发现RA ST中表达程度不同的CitP，而OA ST中未见CitP表达。RA ST中PAD4的表达量明显高于OA患者，且其表达量与CRP存在呈负相关关系。（2）质谱分析结果表明：1）相对SLE和健康组，RA S-IC中共鉴定出129种差异蛋白。2）相对于OA组，RA ST-IC中鉴定到170种差异蛋白，包括玻连蛋白、桥粒蛋白、组蛋白H2A、热休克蛋白75KDa等。3）RA S-IC中共鉴定出5种CitP：α-1-抗胰蛋白酶、血纤维蛋白溶酶原、富含组氨酸的糖蛋白、丛生蛋白、CD5抗原样蛋白。4）与OA ST比较，RA ST-IC中检测到93种CitP，其中包括：角蛋白、组蛋白H2AX、组蛋白H4、α1-抗糜蛋白酶、78 kDa 葡萄糖调节蛋白等。5）从RA SFs中鉴定出182个瓜氨酸化肽、200个瓜氨酸化位点，来源于83种蛋白。（3）与AMCV-RA、SLE患者和健康对照IC相比，AMCV+RA患者血清AMCV+-ICs能显著刺激RA-FLSs生长。（4）用LncRNA Microarray对RA及对照组FLSs中的lncRNAs进行检测，结果显示，与对照组FLSs相比，RA-FLSs中共有135条 lncRNAs差异表达，其中62条上调，73条下调。定量PCR对其中4条lncRNAs进行了验证。ENST00000483588、ENST00000438399、uc004afb.1和ENST00000452247 诊断RA的ROC曲线下面积分别为0.85，0.92，0.97和0.92。上述研究可望进一步阐述ACPA-IC在RA滑膜炎性损伤中的作用及其机制，为探讨RA新的诊断和治疗分子靶标提供理论和实验依据。