目的: 探索下调中性粒细胞能否缓解胶原诱导关节炎(collagen induced arthritis,CIA)小鼠疾病程度,结合测序分析类风湿关节炎(rheumatoid arthritis,RA)患者中性粒细胞外捕获网(neutrophil extratrapping networks,NETs)形成后的转录组表达差异,为NETs影响RA疾病进展的后续研究提供一定的理论基础。方法: 利用DBA1小鼠构建CIA模型,在二次胶原强化免疫后利用抗Ly6G单克隆抗体敲减CIA小鼠体内中性粒细胞(1次/周),持续干预8周后,处死小鼠采集肺、脾、小鼠后肢进行组织病理学观察;磁珠分选法收集4例RA患者外周血中性粒细胞,DAPI染料对中性粒细胞核进行染色,室温避光静置15 min后,镜下观察中性粒细胞形态特征;利用不同浓度(8 nmol/L、16 nmol/L、32 nmol/L)佛波醇-12-肉豆蔻酸酯-13-乙酸酯(phorbol-12-myristate-13-acetate,PMA)刺激后培养2 h并进行NETs标志物染色,荧光倒置显微镜下观察刺激中性粒细胞形成NETs的最佳浓度;利用PMA激活中性粒细胞后收集样本进行转录组测序,分析RA中性粒细胞NETs形成后的转录组表达变化。结果: 中性粒细胞表达下调部分缓解CIA小鼠关节滑膜、肺及脾的病理改变;8 nmol/L PMA刺激RA中性粒细胞放置2 h、37 ℃、5 % CO2培养箱中形成NETs的形态最佳;差异分析筛选出2 742个差异基因(P≤0.05),其中包括1 579个上调差异基因[log2(RA_control/RA_PMA＞0)]和1 163个下调差异基因[log2(RA_control/RA_PMA＜0],从中筛选出340个显著差异基因(log2|RA_control/RA_PMA|≥2),进行PPI网络构建进行展示;340个差异基因的GO、KEGG和GSEA KEGG通路分析分别富集较多为免疫回应和信号受体活性的调控、细胞因子之间相互调控通路、细胞因子受体互作、凋亡和Toll受体信号通路中。结论: 下调中性粒细胞可缓解CIA小鼠关节滑膜、肺及脾的病理改变;RA的NETs形成与免疫调控、凋亡以及细胞因子之间相互作用通路相关,表明NETs的形成可能参与以上免疫过程,对RA疾病进展起重要的作用。
Objective: To explore whether down-regulation of neutrophils can alleviate the disease severity of collagen-induced arthritis (CIA) mice, and to analyze the differences in transcriptome expression after the formation of neutrophil extratrapping networks (NETs), providing a theoretical basis for the follow-up research on the effect of NETs on RA disease progression. Methods: DBA1 mice were used to construct a CIA model, and anti-Ly6G monoclonal antibody was used to knock down neutrophils in CIA mice after secondary collagen boosting immunization (once a week), and the intervention was continued for 8 weeks. After 8 weeks, the mice were sacrificed to collect the lungs, spleens, and hindlimbs of mice for histopathological observation; RA peripheral blood neutrophils were collected by magnetic bead sorting, and neutrophil nuclei were stained with DAPI dye, and kept at room temperature. After standing under light for 15 min, the morphological characteristics of neutrophils were observed under microscope; RA peripheral blood neutrophils were collected, stimulated with different concentrations (8 nmol/L, 16 nmol/L, 32 nmol/L) of PMA, cultured for 2 h, and stained with NETs markers. The optimal concentration of stimulating neutrophils to form NETs was observed under a fluorescent inverted microscope; Samples were collected for transcriptome sequencing after stimulation of neutrophils using PMA to analyze changes in transcriptome expression after formation of RA NETs. Results: Down-regulation of neutrophil expression partially alleviated the pathological changes in the joint synovium, lung and spleen of CIA mice; 8 nmol/L PMA stimulated RA neutrophils for 2 h, 37°C, 5 % CO2 culture; Differential analysis screened out 2742 differential genes(P≤0.05),including 1579 up-regulated differential genes [log2(RA_control/RA_PMA＞0)] and 1163 down-regulated differential genes [log2(RA_control/RA_PMA＜0]. 340 significantly different genes (log2|RA_control/RA_PMA|≥2) were identified and displayed by PPI network construction; GO, KEGG and GSEA KEGG pathway analysis of 340 differential genes were enriched respectively as follows: immune response and signal receptor activity In the regulation of cytokines, the mutual regulation pathway between cytokines, the interaction of cytokine receptors, apoptosis and Toll-like receptor signaling pathways. Conclusion: Down-regulation of neutrophils can alleviate the pathological changes of joint synovium, lung and spleen in CIA mice; the formation of NETs in RA is related to immune regulation, apoptosis and the interaction between cytokines, indicating that the formation of NETs may be involved in the above The immune process plays an important role in the progression of RA disease.
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