oxLDL/β2GPI/抗β2GPI抗体复合物促进A7r5细胞钙化及炎症因子表达  

OxLDL/β2GPI/anti-β2GPI complex promotes calcification and inflammatory cytokine expression in A7r5 cells

作　　者：姚雨叶 蔡谦谦 周红 何超 张鹏 吴倩倩 张贵婷 匡铭 陈芋丹 YAO Yuye;CAI Qianqian;ZHOU Hong;HE Chao;ZHANG Peng;WU Qianqian;ZHANG Guiting;KUANG Ming;CHEN Yudan(Institute of Hematology,School of Medicine,Jiangsu University,Zhenjiang 212013;Department of Laboratory Medicine,Changzheng Hospital,Navy Military Medical University,Shanghai 200003,China)

机构地区：[1]江苏大学医学院血液研究所,江苏镇江212013 [2]海军军医大学附属长征医院实验诊断科,上海200003

出　　处：《细胞与分子免疫学杂志》2019年第12期1069-1075,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81370614)。

摘　　要：目的探讨氧化低密度脂蛋白/β2糖蛋白I/抗β2糖蛋白I抗体(oxLDL/β2GPI/抗β2GPI抗体)复合物对大鼠血管平滑肌A7r5细胞钙化、炎症因子表达的影响以及Toll样受体4(TLR4)在其中的作用。方法分别用oxLDL、oxLDL/β2GPI复合物、β2GPI/抗β2GPI抗体复合物、oxLDL/抗β2GPI抗体复合物、oxLDL/β2GPI/抗β2GPI抗体复合物干预刺激大鼠A7r5细胞系不同时间,采用或不采用TLR4抑制剂TAK-242处理。茜素红染色观察细胞钙化结节,实时荧光定量PCR和Western blot法检测平滑肌蛋白22α(SM22α)和RUNX家族转录因子2(RUNX2)的mRNA和蛋白水平,ELISA检测细胞培养上清中肿瘤坏死因子α(TNF-α)水平;A7r5细胞给予TNF-α刺激后,进一步检测SM22α和RUNX2 mRNA和蛋白水平的变化。结果oxLDL/β2GPI/抗β2GPI抗体复合物能够促进A7r5细胞钙化结节增多,SM22α表达减少而RUNX2明显上升,促进炎症因子TNF-α的表达,TLR4抑制剂能够缓解这一现象;外加不同浓度的TNF-α能够使A7r5细胞SM22α表达下降,RUNX2表达增加。结论oxLDL/β2GPI/抗β2GPI抗体复合物促使A7r5细胞表达炎症因子TNF-α增加,并由此促进细胞发生钙化,同时使细胞由收缩表型向成骨表型发生转变,TLR4参与这一过程。Objective To evaluate the effect of oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2 glycoprotein I antibody(oxLDL/β2GPI/anti-β2GPI)complex on the calcification and inflammatory cytokine expression in A7r5 rat vascular smooth muscle cells,and to find out the role of Toll-like receptor 4(TLR4)signal in this process.Methods The A7r5 cells were intervened with oxLDL,oxLDL/β2GPI complex,oxLDL/anti-β2GPI complex,β2GPI/anti-β2GPI complex,and oxLDL/β2GPI/anti-β2GPI complex for different time,with or without TLR4 inhibitor(TAK-242).Alizarin red staining was used to observe the calcification.Real-time quantitative PCR was applied to detect the total mRNA levels of actin-associated protein SM22α(trangelin)and Runt-related transcription factor 2(RUNX2).The protein levels of SM22αand RUNX2 were measured by Western blotting.Tumor necrosis factorα(TNF-α)was detected by ELISA.Different concentrations of TNF-αwere adopted to stimulate A7r5 cells,and the above methods were operated to examine the changes of SM22 and RUNX2.Results When A7r5 cells were incubated by oxLDL/β2GPI/anti-β2GPI complex,more calcified nodules were observed,the expression of SM22αwas reduced and RUNX2 expression was enhanced at both mRNA and protein levels.And the complex increased the expression of inflammation cytokine TNF-α.TLR4 inhibitor TAK-242 reversed the phenomenon.Different concentrations of TNF-αcould reduce mRNA and protein expression of SM22αwhile raise RUNX2 expression.Conclusion oxLDL/β2GPI/anti-β2GPI complex can increase the expression of cytokine TNF-α,and thus quicken calcification procedure in A7r5 cells,along with the change of the traditional contraction phenotype into the osteogenic phenotype.TLR4 receptor participates in this process.

关 键 词：oxLDL/β2GPI/抗β2GPI抗体复合物 血管平滑肌细胞 肿瘤坏死因子(TNF-α) 血管钙化 

分 类 号：R392.12[医药卫生—免疫学] R364.5[医药卫生—基础医学]