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

摘要

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100 μg/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100 μg/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).100 μg/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 - kB ) 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 - kB p65 protein in cytoplasm and nucleus was detected by Western blotting, representing the activation of NF - kB 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 - kB 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 - KBp65 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 - kB and may play an important role in the beginning of joint inflammation, angio-genesis and bone destruction.