中性粒细胞胞外陷阱对类风湿关节炎成纤维化样滑膜细胞mRNA表达谱的影响

Effect of neutrophil extracellular traps on mRNA expression profiling of fibroblast-like synoviocytes in rheumatoid arthritis patients

目的中性粒细胞胞外陷阱(NETs)与RA疾病的发生发展密切相关。文章旨在研究NETs对RA成纤维化样滑膜细胞(FLS)基因表达谱的影响。方法收集RA患者8例以及8例健康志愿者外周血5 mL分别作为RA组和对照组。分离RA患者与健康对照者的外周血中性粒细胞,免疫荧光法检测NETs的生成。同时ELISA检测RA组与对照组血清中游离核小体的水平。体外分离健康供者的中性粒细胞,以PMA诱导NETs生成。将FLS以1×10~6的数量接种在6孔板中,以含10%FBS的DMEM培养基培养,加或不加所分离的NETs刺激,分为未刺激的FLS组和NETs刺激后的FLS组。全基因组分析NETs对RA FLS基因表达谱的影响。结果与对照组外周血中NETs形成数量(6.67±1.45)相比,RA组(18.67±2.33)显著增多(P<0.05);与对照组(0.11±0.02)相比,RA组血清中NETs组分游离核小体的水平(0.20±0.02)也显著升高(P<0.05)。基因芯片结果进一步提示与未刺激的FLS对比,NETs刺激培养后的FLS共有差异表达mRNA469个,其中mRNA上调257个,下调212个。全基因组学分析显示NETs可以作用于RA FLS,上调炎性反应、细胞增殖、细胞迁移、蛋白酶等相关基因的表达,下调细胞程序性死亡、细胞生长阻滞、DNA损伤相关基因的表达。结论NETs诱导RA FLS的侵袭表型和炎症反应,NETs可通过影响FLS的生物学功能参与RA滑膜炎。

Objective Synovitis is the main pathological feature of rheumatoid arthritis(RA). Recent studies have shown that neutrophil extracellular traps(NETs) are closely related to the occurrence and development of RA. The purpose of this study was to investigate the effect of neutrophil extracellular traps(NETs) on the gene expression profiling of fibroblast-like synoviocytes(FLS) in rheumatoid arthritis(RA). Methods Peripheral blood 5 mL from 8 RA patients and 8 healthy volunteers were collected as RA group and control group, respectively. Neutrophils were isolated from peripheral blood of RA patients and healthy controls. NETs were detected by immunofluorescence assay. At the same time, the levels of free nucleosomes in serum of RA group and control group were detected by ELISA. Neutrophils from healthy donors were isolated in vitro and NETs were induced by PMA. FLS were seeded in a 6-well plate at a quantity of 1×10~6 and cultured in DMEM medium containing 10% FBS with or without the stimulation of isolated NETs. The FLS were divided into unstimulated FLS group and NETs stimulated FLS group. The effect of NETs on RA FLS gene expression profiling was analyzed by genome-wide analysis. Results Compared with the control group(6.67±1.45), the number of NETs in the RA group(18.67±2.33) was significantly increased(P<0.05);Compared with the control group(0.11±0.02), the free nucleosome level in serum of NETs components in RA group(0.20±0.02) was also significantly increased(P<0.05). Microarray results further indicated that compared with unstimulated FLS, 469 mRNAs were differentially expressed in NETs stimulated FLS, including 257 up-regulated mRNAs and 212 down-regulated mRNAs. Genome-wide analysis showed that NETs could act on RA FLS, up-regulate the expression of inflammatory response, cell proliferation, cell migration, protease and other related genes, and down-regulate the expression of genes related to programmed cell death, cell growth arrest, and DNA damage. Conclusion NETs can induce the invasive phenotype and inflammatory response of RA FLS. NETs may be involved in RA synovitis by affecting the biological function of FLS.