中性粒细胞胞外诱捕网在类风湿关节炎致病机制中的初步探究

A preliminary study on the pathogenesis of neutrophil extracellular traps in rheumatoid arthritis

目的探究中性粒细胞胞外诱捕网(NETs)对类风湿关节炎滑膜成纤维细胞(RA-FLSs)的潜在作用。方法取RA患者滑膜组织分离培养;提取健康人外周血中性粒细胞,刺激NETs形成并提取NETs;用CellTiter96 AQueous单溶液试剂(MTS)增殖实验评估NETs对RA-FLSs增殖的影响;实时荧光定量逆转录聚合酶链反应(qRT-PCR)测定NETs持续刺激RA-FLSs60h后细胞中结缔组织生长因子(CTGF)mRNA的表达情况。采用配对样本t检验和单因素方差分析对结果进行处理。结果分离纯化的中性粒细胞体外刺激可形成NETs,提取的NETs测定DNA浓度为58.5ng/μl(1×10^6细胞);与对照组(0μlNETs)相比,NETs能促进RA-FLSs增殖,且随着NETs浓度增加其促进RA-FLSs增殖的能力也增强(F=99.519,P<0.01);与对照组(0μlNETs)相比,10μlNETs可以明显促进RA-FLSs增殖(t=-12.226,P<0.01);用脱氧核糖核酸酶Ⅰ(DNaseⅠ)预处理NETs后,其促进RA-FLSs增殖的作用受到抑制(t=-2.376,P=0.049);NETs能刺激RA-FLSs内CTGFmRNA表达上调[(30.7±0.5),t=12.13,P<0.01]。结论NETs在体外能促进RA-FLSs增殖并刺激RA-FLSs内CTGFmRNA表达上调。

Objective To explore the potential effects of neutrophil extracellular traps (NETs) on rheumatoid arthritis synovial fibroblasts (RA-FLSs). Methods The synovial tissues of RA patients were isolated and cultured in vitro. Peripheral blood neutrophils were extracted from healthy volunteers and used to stimulate NETs' formation, following with NETs' extraction. MTS proliferation assay was used to evaluate the effect of NETs on the proliferation of RA-FLSs. QRT-polymerase chain reaction (q-PCR) was used to determine the expression of connective tissue growth factor (CTGF) mRNA in cells treated with NETs-stimulated RA-FLSs for 60 h. The results were processed using paired sample t-test and one-way analysis of variance (ANOVA). Results The isolated and purified neutrophils could form NETs by in vitro stimulation. The concentration of extracted NETs-DNA was 58.5 ng/μl (1×10^6 cells). Compared with the control group (0 μl NETs), NETs could promote the proliferation of RA-FLSs. With the increase of NETs' concentration, the proliferation of RA-FLSs was also enhanced (F=99.519, P<0.05). Compared with the control group (0 μl NETs), 10 μl NETs could signi-ficantly promote the proliferation of RA-FLSs (t=-12.226, P<0.01). Pretreatment of NETs with DNase Ⅰ inhi-bited its effect on promoting the proliferation of RA-FLSs (t=-2.376, P=0.049), NETs stimulated the up-re-gulation of CTGF mRNA expression in RA-FLSs [(30.7±0.5), t=12.13, P<0.01]. Conclusion NETs can pro-mote the proliferation of RA-FLSs and stimulate the up-regulation of CTGF mRNA in RA-FLSs in vitro.