Department of Rheumatology, Third Hospital Affiliated to Sun Yat-sen University, No. 600 Tianhe Road, Guangzhou, China.
Int J Rheum Dis. 2012 Jun;15(3):322-9. doi: 10.1111/j.1756-185X.2012.01721.x. Epub 2012 Feb 28.
After the onset of rheumatoid arthritis (RA), fibroblast-like synoviocytes (RA-FLS) which are specialized types of fibroblasts, become tumor-like, keeping their ability to increase proliferation and invasion. The mechanism of their tumor-like growth is unclear. Fractalkine (FKN), also called CX3CL1, plays an important role in the proliferation of cells. FKN may stimulate the proliferation of RA-FLS and the by nuclear factor κB (NF-κB) pathway may be one of the steps in this process.
To investigate whether FKN can stimulate cell growth and increase its expression in RA-FLS, and the relationship between the NF-κB pathway and the function of FKN.
FLS were isolated from primary synovial tissue obtained from three patients with RA who had undergone total joint replacement surgery or synovectomy in the Third Hospital Affiliated to Sun Yat-sen University from February 2009 to January 2010. FKN was used in different concentrations to stimulate RA-FLS with or without NF-κB pathway blocker (PDTC), and to test the proliferation of FLS after 24 h by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RA-FLS was treated with 100 ng/mL FKN or 100 μM PDTC for different periods, and messenger RNA (mRNA) expression of FKN and CX3CR1 in RA-FLS was detected by reverse transcription - polymerase chain reaction. We then tested the protein expression of NF-κBp65 in the cytoplasm and nucleus, respectively by Western blotting after treating the RA-FLS with 100 ng/mL FKN for different time periods.
FKN stimulated cell growth in RA-FLS at the concentration of 50 or 100 ng/mL (P = 0.005 and P = 0.022, respectively). NF-κB pathway blocker inhibited FKN, promoting proliferation of RA-FLS. RA-FLS could express FKN and CX3CR1 mRNA in vitro. FKN up-regulated FKN expression after 18-h treatment (P = 0.012). PDTC disturbed the expression of FKN mRNA after 16-18 h treatment (P = 0.001 and P < 0.001, respectively). After stimulation with FKN for 1 h, the expression of NF-κBp65 in cytoplasm began to decrease (P = 0.010), and the expression of NF-κBp65 in the nucleus began to increase after 2 h (P = 0.011).
These results suggest that FKN stimulates cells growth in RA-FLS and NF-κB pathway blocker inhibits FKN, promoting proliferation of RA-FLS. FKN induced activation of NF-κB activity. FKN up-regulates FKN mRNA expression in RA-FLS via the NF-κB pathway.
类风湿关节炎（RA）发病后，成纤维样滑膜细胞（RA-FLS）成为肿瘤样细胞，保持增殖和侵袭能力。其肿瘤样生长的机制尚不清楚。趋化因子（FKN）又称 CX3CL1，在细胞增殖中起重要作用。FKN 可能刺激 RA-FLS 的增殖，核因子 κB（NF-κB）途径可能是这一过程中的步骤之一。
探讨 FKN 是否能刺激 RA-FLS 细胞生长并增加其表达，以及 NF-κB 途径与 FKN 功能的关系。
2009 年 2 月至 2010 年 1 月，中山大学附属第三医院对 3 例接受全关节置换术或滑膜切除术的 RA 患者的原发性滑膜组织进行分离，获得成纤维样滑膜细胞。用不同浓度的 FKN 刺激 RA-FLS，或用 NF-κB 途径阻滞剂（PDTC）处理，用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法检测 24 h 后 FLS 的增殖情况。用 100ng/ml FKN 或 100μM PDTC 处理 RA-FLS 不同时间，用逆转录-聚合酶链反应检测 RA-FLS 中 FKN 和 CX3CR1 的信使 RNA（mRNA）表达。然后用 Western blot 法分别检测 100ng/ml FKN 处理不同时间后 RA-FLS 胞质和核内 NF-κBp65 的蛋白表达。
FKN 在 50 或 100ng/ml 浓度下刺激 RA-FLS 细胞生长（P=0.005 和 P=0.022）。NF-κB 途径阻滞剂抑制 FKN，促进 RA-FLS 增殖。RA-FLS 可在体外表达 FKN 和 CX3CR1 mRNA。FKN 处理 18 h 后上调 FKN 表达（P=0.012）。PDTC 处理 16-18 h 后干扰 FKN mRNA 表达（P=0.001 和 P<0.001）。FKN 刺激 1 h 后，胞质中 NF-κBp65 的表达开始下降（P=0.010），2 h 后核内 NF-κBp65 的表达开始增加（P=0.011）。
这些结果提示 FKN 刺激 RA-FLS 细胞生长，NF-κB 途径阻滞剂抑制 FKN 促进 RA-FLS 增殖。FKN 诱导 NF-κB 活性激活。FKN 通过 NF-κB 途径上调 RA-FLS 中 FKN mRNA 的表达。
