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.
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)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
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)。
