oxLDL/β2GPI/抗β2GPI抗体复合物促进A7r5细胞趋化和迁移及脂质累积

The oxLDL/β2GPI/anti-β2GPI antibody complex promotes A7r5 chemotaxis,migration and lipid accumulation

目的探讨氧化型低密度脂蛋白/β2糖蛋白I/抗β2糖蛋白I抗体(oxLDL/β2GPI/β2GPI-Ab)复合物对A7r5大鼠胸主动脉平滑肌细胞迁移、趋化和脂质累积的影响,以及Toll样受体4(TLR4)在其中的作用。方法体外传代细胞株,分别以oxLDL、oxLDL/β2GPI复合物、β2GPI、抗β2GPI抗体、β2GPI/β2GPI-Ab复合物、oxLDL/β2GPI/β2GPI-Ab复合物刺激经TLR4阻断剂TAK-242预处理和未经预处理的A7r5细胞。提取细胞总RNA,实时荧光定量PCR检测单核细胞趋化蛋白1(MCP-1)、基质金属蛋白酶9(MMP-9)、乙酰辅酶A乙酰基转移酶1(ACAT1)的mRNA水平;收集培养上清,ELISA检测细胞上清液中MCP-1、MMP-9的蛋白水平;划痕实验观察A7r5细胞的迁移。以oxLDL、oxLDL/β2GPI/β2GPI-Ab复合物刺激经TAK-242预处理和未经处理的A7r5细胞,用胆固醇试剂盒检测细胞内总胆固醇(TC)、游离胆固醇(FC)含量并计算胆固醇酯(CE)的含量。结果oxLDL/β2GPI/β2GPI-Ab复合物促进A7r5细胞的迁移,加速胞内胆固醇的累积,促进MCP-1、MMP-9、ACAT1的表达,而TLR4阻断剂TAK-242能够抑制上述现象。结论 oxLDL/β2GPI/β2GPI-Ab复合物能经TLR4依赖的方式促进A7r5细胞趋化、迁移和脂质累积,参与动脉粥样硬化进程。

Objective To explore the effect of the oxidized low-density lipoprotein/β2-glycoprotein I/β2 glycoprotein I antibody（ oxLDL/β2 GPI/β2 GPI-Ab） complex on the migration,chemotaxis and lipid accumulation of rat thoracic aortic smooth muscle A7 r5 cell line,and unveil the role of Toll-like receptor 4（ TLR4） pathway during the process. Methods A7 r5 cells were cultured in vitro with or without the pretreatment of TAK-242,a TLR4 inhibitor. Then the cells were stimulated by oxLDL,oxLDL/β2 GPI complex,β2 GPI,β2 GPI-Ab,β2 GPI/β2 GPI-Ab complex or oxLDL/β2 GPI/β2 GPI-Ab complex. Total RNA was extracted from the cel s and the mRNA levels of monocyte chemoattractant protein-1（ MCP-1）,matrix metal oproteinase-9（ MMP-9） and acetyl-Co A acetyltransferase 1（ ACAT1） were evaluated by real-time quantitative PCR. The cell supernatants were collected,and the protein levels of MCP-1 and MMP-9 were determined by ELISA. The migration of A7 r5 was observed through wound-healing test. A7 r5 cel s pretreated with or without TAK-242 were stimulated by oxLDL or oxLDL/β2 GPI/β2 GPI-Ab complex,and the content of total cholesterol（ TC） and free cholesterol（ FC） in them were measured by corresponding test kits. Then the level of intracellular cholesterol ester（ CE） was calculated. Results The oxLDL/β2 GPI/β2 GPI-Ab complex promoted the migration and cholesterol accumulation,and up-regulated the expressions of MCP-1,MMP-9 and ACAT1 in A7 r5 cells. In addition,the application of TLR4 inhibitor,TAK-242,suppressed these effects. Conclusion oxLDL/β2 GPI/β2 GPI-Ab complex contributes to the process of atherosclerosis through promoting the migration,chemotaxis and lipid accumulation of a7 r5 in a TLR4-dependent manner.