联胺对内皮细胞表达巨噬细胞炎性蛋白-1α的影响

The effect of diamide on the expression of macrophage inflammatory protein-1α in endothelial cells

目的 观察联胺能否诱导培养的人脐静脉内皮细胞表达和分泌巨噬细胞炎性蛋白 1α(MIP 1α)及其意义。方法 使内皮细胞暴露于不同浓度联胺 4h ,以核酸酶S1保护分析法检测内皮细胞内MIP 1αmRNA ,以细胞酶联免疫吸附实验测定内皮细胞的MIP 1α蛋白表达。同时收集内皮细胞条件培养基 ,用Boyden小室微孔滤膜法检测MIP 1α对外周血单核细胞的趋化活性。结果 内皮细胞MIP 1αmRNA在 5 μmol/L联胺组的表达是对照组的 3 4倍 ,差异具有显著性 (t=8 70 ,P <0 0 5 )。1、5、10 μmol/L联胺组MIP 1α蛋白的表达分别是对照组的 1 9倍、2 2倍、1 7倍 ,方差分析显示有统计学意义 (F =35 6 5 ,P <0 0 5 )。趋化实验显示 ,5 μmol/L联胺组内皮细胞的条件培养基引起单核细胞的迁移距离 [(99 5 0± 4 31) μm]显著高于无联胺组 [(6 6 47± 3 2 5 ) μm]、化学促动组 [(6 7 0 3± 6 83)μm]和随机移动组 [(6 5 40± 3 36 ) μm ,F =40 4 31,P <0 0 5 ],提示经联胺刺激的条件培养基内含有具趋化活性的物质。加入山羊抗人MIP 1α多克隆抗体后 ,联胺组条件培养基所致的单核细胞迁移距离降至 (82 80± 6 88) μm(F =192 2 5 ,P <0 0 5 ) ,说明经联胺刺激的条件培养基内含有具趋化活性的MIP 1α。结论 脂质过氧?

Objective To study the effect of diamide on the expression of macrophage inflammatory protein-1α (MIP-1α) in cultured human umbilical vein endothelial cells. Methods After exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1α mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1α protein in those cells was determined by cell enzyme-linked immunosorbent assay . The chemotactic activity of MIP-1α in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers. Results Incubation of ECs with 5 μmol/L diamide resulted in a 2.4-fold increase in the level of MIP-1α mRNA expression as compared with the control group (t=8.70, P<0.05). Exposure of ECs to 1 μmol/L, 5 μmol/L and 10 μmol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1α protein expression respectively, as compared with the control group (F=35.65, P<0.05). Chemotactic assay showed that the migration distance of monocytes towards the conditioned medium (CM) of ECs treated with 5 μmol/L diamide was 99.50 μm±4.31 μm, which was significantly more than the 66.47 μm±3.25 μm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 μm±6.83 μm) and the random migration group (65.40 μm±3.36 μm) (F=404.31, P<0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 μmol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 μmol/L diamide was significantly reduced to 82.80 μm±6.88 μm after the addition of goat anti-human MIP-1α antibody (F=192.25, P<0.05), which indicates the chemotactic activity of MIP-1α in the conditioned medium of the ECs in the diamide group. Conclusions Diamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1α with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.