抗环瓜氨酸化抗体分离及其对巨噬细胞功能影响
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摘要
从类风湿性关节炎(RA)患者血清中分离抗环瓜氨酸化抗体(ACPAs),观察ACPAs对巨噬细胞功能的影响,探讨ACPAs影响RA病情发展的可能机制。利用亲和层析从RA患者血清中分离ACPAs,通过荧光定量PCR、蛋白印迹(Western blotting)及流式细胞术等实验方法观察ACPAs对巨噬细胞功能的影响。结果表明:亲和层析可很好地从血清中分离ACPA。ACPAs(40 000 IU/L)可促进巨噬细胞肿瘤坏死因子α(TNFα)、白细胞介素6(IL-6)、诱导型一氧化氮合成酶(iNOS)、人类白细胞DR抗原(HLA-DR)、磷酸化蛋白激酶B(p-AKT)等炎性因子的表达。SiRNA转染实验证实:ACPAs通过促进AKT蛋白磷酸化,诱导TNFα、IL-6、iNOS的表达。
Anti-citrullinated protein antibodies(ACPAs)were isolated from rheumatoid arthritis(RA)patient,and the mechanism of ACPAs-induced macrophage activities was studied.ACPAs were purified by affinity column.Fluorescence-activated cell sorting,Western blotting,and real-time PCR analyses were performed to detect the ACPAs-induced macrophage action.Results show that ACPAs(40 000 IU/L)could induce tumor necrosis factor α(TNFα),interleukin(IL)-6,inducible nitric oxide synthase(iNOS),human leukocyte antigen(HLA)-DR and phosphorylated protein kinase B(p-AKT)expression in macrophage.ACPAs increased TNFα,IL-6 and iNOS expression through promoting p-AKT expression.
Anti-citrullinated protein antibodies(ACPAs)were isolated from rheumatoid arthritis(RA)patient,and the mechanism of ACPAs-induced macrophage activities was studied.ACPAs were purified by affinity column.Fluorescence-activated cell sorting,Western blotting,and real-time PCR analyses were performed to detect the ACPAs-induced macrophage action.Results show that ACPAs(40 000 IU/L)could induce tumor necrosis factor α(TNFα),interleukin(IL)-6,inducible nitric oxide synthase(iNOS),human leukocyte antigen(HLA)-DR and phosphorylated protein kinase B(p-AKT)expression in macrophage.ACPAs increased TNFα,IL-6 and iNOS expression through promoting p-AKT expression.
引文
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[2] DEANE K D.Rheumatoid arthritis:autoantibodies,citrullinated histones and initiation of synovitis[J].Nat Rev Rheumatol,2015,11(12):688-689.
[3] BURMESTER G R,FEIST E,D?RNER T.Emerging cell and cytokine targets in rheumatoid arthritis[J].Nat Rev Rheumatol,2014,10(2):77-88.
[4] CHEUNG T T,MCINNES I B.Future therapeutic targets in rheumatoid arthritis[J].Semin Immunopathol,2017,39(4):487-500.
[5] HORWOOD N J.Macrophage polarization and bone formation:a review[J].Clin Rev Allergy Immunol,2016,51(1):79-86.
[6] LI C,XU M M,WANG K,et al.Macrophage polarization and meta-inflammation[J].Transl Res,2018,191:29-44.
[7] LU M C,LAI N S,YU H C,et al.Anti-citrullinated protein antibodies bind surface-expressed citrullinated GRP78 on monocyte/macrophages and stimulate tumor necrosis factor α production[J].Arthritis Rheum,2010,62:1213-1223.
[8] LU M C,LAI N S,YIN W Y,et al.Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells[J].J Clin Immunol,2013,33:558-566.
[9] CLAVEL C,NOGUEIRA L,LAURENT L,et al.Induction of macrophage secretion of tumor necrosis factor alpha through Fcγ receptor Ⅱa engagement by rheumatoid arthritis-specific autoantibodies to citrullinated proteins complexed with fibrinogen[J].Arthritis Rheum,2008,58(3):678-688.
[10] SOKOLOVE J,ZHAO X,CHANDRA P E,et al.Immune complexes containing citrullinated fibrinogen co-stimulate macrophages via Toll-like receptor 4 and Fcγ receptor[J].Arthritis Rheum,2011,63(1):53-62.