氧化低密度脂蛋白对巨噬细胞的作用及机制

Experimental study of the effect of oxidized low density lipoprotein on macrophages

目的 观察氧化低密度脂蛋白（oxLDL）对巨噬细胞增殖和分泌巨噬细胞迁移抑制因子（MMIF/MIF）的作用.方法 （1）制备人单核细胞来源巨噬细胞（MDMs）,25、50和75 mg/L浓度oxLDL培养12、24、48 h后,细胞计数试剂盒（CCK-8）法检测细胞增殖,酶联免疫吸附法测定上清MIF浓度;不含oxLDL培养为正常对照（NC）组.（2）制备含稳转核因子-κB （NF-κB）-LUC质粒的MDMs,同法培养,荧光素酶底物（Luciferase）检测细胞NF-κB通路活性.（3）含NF-κB-LUC质粒的MDMs中加入终质量浓度为0、10 μmol/L的NF-κB通路抑制剂BAY 11-7082,同法培养并检测上清MIF浓度.结果 （1）与NC组比较,25、50、75 mg/L oxLDL组12 h时MDMs增殖显著升高,48 h时分别达到17.7％、27.8％和41.2％ （P ＜0.01）;其上清液MIF也在12h起升高,48 h时分别是NC组的1.49、1.67和2.09倍（P＜0.01）.（2）与NC组比较,oxLDL各组在12h即可检测到NF-κB通路明显激活,24h时MDMs内NF-κB通路活性分别增加38.1％、60.3％和61.1％ （P＜0.01）.（3）加入BAY11-7082后,各组上清液MIF浓度在12 h即出现明显下降,48 h时分别下降58.6％、69.3％、69.7％和73.5％ （P＜0.01）.结论 25 ～ 75 mg/L浓度的oxLDL可促进MDMs增殖和MIF的分泌,其作用随oxLDL作用时间的延长和浓度的增高而增强.oxLDL可能通过诱导激活NF-κB信号通路促进MDMs合成分泌MIF.

Objective To investigate the effect of oxidized low density lipoprotein （oxLDL） on macrophages and their secretion of macrophage migration inhibitory factor （MMIF/MIF）.Methods （1）Preparation of monocytes derived macrophages （MDMs）,co-culture with oxLDL at concentrations of 25,50,75 mg/L,respectively.MDMs cultured without oxLDL were normal controls （NC）.MDM proliferation was detected using cell counting kit-8 assay （CCK-8 assay） ; supernatant MIF was examined using enzyme linked immunosorbent assay （ELISA）.（2） Preparation of MDMs derived from THP-1 cell lines which were transfected with nuclear factor-κB （NF-κB）-LUC plasmids,co-culture with oxLDL as above-mentioned,NF-κB pathway activity was tested by luciferase test.（3） MDMs containing NF-κB-LUC plasmids were co-cultured with oxLDL,self-control study was performed at the same concentration of oxLDL,with or without adding NF-κB pathway inhibitor BAY 11-7082 at the concentration of 10 μmol/L to culture medium.Supernatant MIF was detected.Results （1） Compared with NC,significantly increased MDM proliferation occurred 12 hours after co-culture with oxLDL,the proliferation increased by 17.7%,27.8% and 41.2%,respectively at 48 hours （P 〈 0.01） ; supernatant MIF level increased simultaneously,reaching 1.49,1.67 and 2.09 folds,respectively at 48 hours,as compared with NC （P〈0.01）.（2）Compared with NC,significant activation of NF-κB pathway was detected 12 hours after co-culture with oxLDL,the activation was increased by 38.1%,60.3% and 61.1%,respectively at 48 hours （P 〈0.01）.（3） Compared with NF-κB pathway inhibitor BAY 11-7082-free group,supematant MIF level decreased significantly at 12 hours in BAY 11-7082 group,it decreased by 58.6%,69.3%,69.7% and 73.5%,respectively at 48 hours （P 〈 0.01）.Conclusion oxLDL at concentrations of 25 to 75 mg/L could promote MDM proliferation and MIF secretion,this effect was enhanced with prolonged co-culture time.The increased MIF secretion induced by oxLDL might be achieved through activation of the NF-κB pathway.