Fractalkine对类风湿关节炎患者成纤维样滑膜细胞中NF-κB活化及内源性fractalkine mRNA表达的影响

Effects of fractalkine on nuclear factor-κB activation and endogenous fractalkine mRNA expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis

目的:观察Fractalkine(FKN)对类风湿关节炎(RA)患者成纤维样滑膜细胞(FLS)核转录因子κB(NF-κB)活化以及内源性FKN mRNA表达的影响。方法:通过组织块法培养RA-FLS。在RA-FLS中加入100μg/L FKN,分别作用0 h、1 h及2 h,用Western blotting检测RA-FLS胞浆及胞核中NF-κB p65蛋白表达量变化以明确NF-κB的活化情况;加入100μg/L重组人FKN分别作用0 h、12 h、18 h后,用RT-PCR检测FKN mRNA表达的变化。结果:重组人FKN刺激1 h后,RA-FLS胞浆中NF-κB p65蛋白水平明显低于无FKN刺激的对照组(P<0.05);FKN刺激后2 h,细胞核中NF-κB p65蛋白水平较对照组明显升高(P<0.05)。100μg/L重组人FKN刺激RA-FLS,呈时间依赖方式诱导内源性FKN mRNA表达。FKN刺激RA-FLS18 h,细胞中FKN mRNA的表达水平明显升高(P<0.05)。结论:外源FKN可刺激RA-FLS内源性的FKN mRNA表达上升,提示RA-FLS中可能存在FKN的正反馈现象。FKN对NF-κB有活化作用,可能对RA患者关节中炎症的启动、血管生成和骨质破坏有重要作用。

AIM：To investigate the effects of fractalkine（FKN） on nuclear factor kappa B（NF-κB） activation and endogenous FKN mRNA expression in fibroblast-like synoviocytes（FLS） from the patients with rheumatoid arthritis（RA）.METHODS： RA-FLS were gained through tissue culture.Fractalkine at 100 μg/L was used to stimulate RA-FLS for 0 h,1 h and 2 h.The expression of NF-κB p65 protein in cytoplasm and nucleus was detected by Western blotting,representing the activation of NF-κB in RA-FLS.RA-FLS was stimulated with fractalkine at concentration of 100 μg/L for 0 h,12 h or 18 h,and the mRNA expression of FKN in RA-FLS was detected by RT-PCR.RESULTS： After stimulated with recombinant human FKN for 1 h,the expression of NF-κB p65 protein in the cytoplasm of RA-FLS was obviously lower than that in RA-FLS without FKN treatment in control group（P〈0.05）.After stimulated with FKN for 2 h,the expression of NF-κBp65 protein in nucleus was obviously higher than that in RA-FLS of control group（P〈0.05）.Recombinant human FKN at concentration of 100 μg/L induced endogenous FKN mRNA expression in RA-FLS in a time-dependent manner.The mRNA expression of FKN in RA-FLS obviously increased after stimulated with FKN for 18 h（P〈0.05）.CONCLUSION： FKN up-regulates the expression of endogenous FKN mRNA,suggesting a positive feedback.FKN can activate the NF-κB and may play an important role in the beginning of joint inflammation,angiogenesis and bone destruction.