RA滑膜成纤维样细胞外泌体促进巨噬细胞向M1极化

M1 polarization is induced by exosomes derived from fibroblast-like synoviocytes from patients with rheumatoid arthritis

摘要:

目的 探讨类风湿关节炎滑膜成纤维样细胞（rheumatoid arthritis fibroblast-like synoviocytes， RA-FLS）外泌体在调控巨噬细胞(macrophage, M)极化中的作用。方法 收集6例骨关节炎（osteoarthritis, OA）患者和6例类风湿关节炎（rheumatoid arthritis， RA）患者关节液和滑膜组织，流式微珠阵列（CBA）法检测关节液中M1和M2型细胞因子的表达；免疫组织化学法检测滑膜中外泌体标志物CD9的表达；分离培养原代RA-FLS和OA-FLS并提取上清液外泌体，使用Western印迹法和粒径分析（nanoparticle tracking analysis, NTA）鉴定；实时荧光定量PCR(RT-PCR）和CBA法分别检测OA-FLS和RA-FLS外泌体对M1和M2型极化分子mRNA水平及细胞因子分泌的影响。结果 RA关节液中M1型细胞因子IL-1β、IL-6、IL-8及M2型细胞因子IL-10水平均显著高于OA组；RA滑膜中CD9高表达；RA-FLS分泌的外泌体较OA-FLS明显增多；RA-FLS外泌体刺激M后，细胞M1型极化分子mRNA表达和细胞因子分泌均增加。结论 RA-FLS外泌体促进M向M1极化。

Abstract:

Objective To investigate the role of exosomes derived from fibroblast-like synoviocytes from patients with rheumatoid arthritis(RA-FLS) in macrophage(M) polarization. Methods Synovial fluid and tissue from osteoarthritis(OA） patients(n=6） and rheumatoid arthritis(RA) patients（n=6）were collected, and the cytokines in joint fluid were detected using flow cytometry bead array(CBA) method. The expression of extracellular marker CD9 in the synovium was detected with immunohi-stochemical method. Primary synovial RA-FLS and OA-FLS were isolated, and the exosomes from cell-culture supernatant were identified by Western blotting and nanoparticle tracking analysis(NTA). The effects of OA-FLS and RA-FLS exosomes on the M1 and M2 phenotype genes were determined with real time fluorescence quantitative PCR(qRT-PCR）and CBA. Results The expressions of M1 type markers（IL-1β, IL-6, IL-8）, and M2 type marker（IL-10） in RA joint fluid were significantly higher than those in the OA group. High expression of CD9 was detected in RA synovium. RA-FLS was found to secrete more exosomes than OA-FLS. Elevated gene and protein expression levels of M1 markers were found in M treated with RA-FLS derived exosomes. Conclusion RA-FLS derived exosomes can induce M1 polarization in vitro.