抗瓜氨酸化肽特异性免疫复合物对类风湿关节滑膜细胞功能影响的研究

The effects of anti-citrullinated peptide antibody specific immune complexes on the functions of fibroblast-like synoviocyte in rheumatoid arthritis

目的:研究类风湿关节炎(RA)患者血清抗瓜氨酸化肽特异性免疫复合物(ACPA-IC)对RA患者关节成纤维样滑膜细胞(FLS)增殖和分泌细胞因子功能的影响。方法:用PEG沉淀法提取RA患者血清免疫复合物(IC),ELISA法检测IC中ACPA-IC水平。用G蛋白免疫亲和层析法从6份ACPA-IC(+)RA血清、4份ACPA-IC(-)RA血清及10份正常人血清中提取IC。体外培养RA关节FLS;分别用RA ACPA-IC(+)提取物、ACPA-IC(-)提取物及健康人血清IC(C-IC)刺激FLS,用CCK-8(Cell Counting Kit-8)试剂检测FLS增殖情况,液态芯片技术检测不同来源IC刺激对FLS分泌IL-1β、IL-2、IL-6、IL-8、IL-10、IL-15、IL-17、TNF-α、GM-CSF、EGF、VEGF等11种细胞因子水平的影响。结果:RA患者ACPA-IC(+)提取物能够显著促进FLS增殖,与对照组IC比较,在培养24小时时间里,四种浓度(25、50、75和100μg/ml)的ACPA-IC都能显著促进FLS增殖。RA患者血清ACPA-IC(+)和ACPA-IC(-)提取物刺激FLS 24小时后,可诱导细胞分泌大量IL-6、IL-8和GM-CSF;其中ACPA-IC(+)组刺激FLS分泌GM-CSF和IL-8量均显著高于ACPA-IC(-)组和C-IC组,ACPA-IC(+)组刺激FLS分泌IL-6水平显著高于C-IC组,但与ACPA-IC(-)组无显著性差异。三组间刺激分泌IL-1β、IL-2、IL-10、IL-15、IL-17、TNF-α、EGF、VEGF等其他8种细胞因子含量均无显著性差异。结论:RA患者血清ACPA-IC可促进FLS增殖,并诱导其分泌IL-6、IL-8和GM-CSF等炎性细胞因子,进而进一步诱发滑膜炎症反应及骨质破坏。

Objective：To study the effects of anti-citrullinated peptide antibody （ACPA） specific immune complexes （ACPA- IC） from rheumatoid arthritis （RA） patients sera on the functions of fibroblast-like synoviocyte. Methods ：The serum IC from RA pa- tients and healthy controls were isolated by polyethylene glycol （PEG） precipitation. The levels of ACPA-IC in the IC were determined by ELISA. fibroblast-like synoviocytes（FLSs） from RA patients were cultivated in vitro and were stimulated with ACPA-IC positive IC （ ACPA-IC（ ＋ ） ） ,ACPA-IC negative IC （ ACPA-IC （ - ） ） of RA patients serum, and healthy control IC （C-IC） respectively, which was extracted by affinity chromatography with immobilized protein G. CCK-8 test and liquichip assay were used to investigate the roles of ACPA-IC on proliferation and seereation of cytokines such as IL-1β, IL-2, IL-6, IL-8, IL-10, IL-15, IL-17, TNF-α, GM-CSF, EGF, VEGF of FLS respectively. Results ： The stimulation of ACPA-IC could notably enhance the proliferation rates of FLSs, compared with the C- IC. The secrection levels of GM-CSF, IL-6 and IL-8 were greatly increased after stimulation of ACPA-IC. The levels of GM-CSF and IL-8 in the supernatants of cells stimulated with ACPA-IC were significantly higher than those of other two groups. The levels of IL-6 in the supernatants of cells stimulated with ACPA-IC （ ＋ ） or ACPA-IC （ - ） were notably higher than C-IC group. However, no signifi- cant differences were detected between ACPA-IC （ ＋ ） and ACPA-IC （ - ） groups. There were no any significant differences of othereight kinds of cytokines after stimulations of ICs. Conclusion： ACPA-IC may induce the proliferation of FLSs and secretions of GM- CSF, IL-6 and IL-8 from FLS, which is responsible for inducing inflammatory response and joint damage.