Yang Limin, Zhu Xuewei, Zhao Xia, Deng Zhongduan
Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Bing Li Xue Za Zhi. 2002 Oct;31(5):427-31.
To study the effect of diamide on the expression of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in cultured human umbilical vein endothelial cells.
After exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1 alpha mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1 alpha protein in those cells was determined by cell enzyme-linked immunosorbent assay. The chemotactic activity of MIP-1 alpha in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers.
Incubation of ECs with 5 micro mol/L diamide resulted in a 2.4-fold increase in the level of MIP-1 alpha mRNA expression as compared with the control group (t = 8.70, P < 0.05). Exposure of ECs to 1 micro mol/L, 5 micro mol/L and 10 micro mol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1 alpha 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 micromol/L diamide was 99.50 microm +/- 4.31 microm, which was significantly more than the 66.47 microm +/- 3.25 microm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 microm +/- 6.83 microm) and the random migration group (65.40 microm +/- 3.36 microm) (F = 404.31, P < 0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 micro mol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 micromol/L diamide was significantly reduced to 82.80 microm +/- 6.88 microm after the addition of goat anti-human MIP-1 alpha antibody (F = 192.25, P < 0.05), which indicates the chemotactic activity of MIP-1 alpha in the conditioned medium of the ECs in the diamide group.
Diamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1 alpha with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.
研究二酰胺对培养的人脐静脉内皮细胞中巨噬细胞炎性蛋白-1α（MIP-1α）表达的影响。
将内皮细胞暴露于不同浓度的二酰胺中4小时后，采用核酸酶S1保护试验检测细胞中的MIP-1α mRNA，并用细胞酶联免疫吸附测定法测定细胞中的MIP-1α蛋白。使用改良的Boyden小室通过微孔滤膜法检测用二酰胺处理的内皮细胞条件培养基中MIP-1α对外周血单核细胞的趋化活性。
与对照组相比，用5 μmol/L二酰胺孵育内皮细胞导致MIP-1α mRNA表达水平增加2.4倍（t = 8.70，P < 0.05）。与对照组相比，将内皮细胞暴露于1 μmol/L、5 μmol/L和10 μmol/L二酰胺中分别导致MIP-1α蛋白表达水平增加0.9倍、1.2倍和0.7倍（F = 35.65，P < 0.05）。趋化试验表明，用5 μmol/L二酰胺处理的内皮细胞条件培养基吸引单核细胞的迁移距离为99.50 μm ± 4.31 μm，显著大于非二酰胺组内皮细胞条件培养基（66.47 μm ± 3.25 μm）、化学动力学组（67.03 μm ± 6.83 μm）和随机迁移组（65.40 μm ± 3.36 μm）（F = 404.31，P < 0.05）。结果显示，5 μmol/L二酰胺处理的内皮细胞条件培养基中可能存在趋化物质。加入山羊抗人MIP-1α抗体后，单核细胞向暴露于5 μmol/L二酰胺的内皮细胞条件培养基的迁移距离显著缩短至82.80 μm ± 6.88 μm（F = 192.25，P < 0.05），这表明二酰胺组内皮细胞条件培养基中MIP-1α具有趋化活性。
二酰胺作为一种脂质过氧化诱导剂，可刺激内皮细胞产生高水平的具有趋化活性的MIP-1α，并可能通过吸引外周血单核细胞进入动脉内膜在动脉粥样硬化形成中起重要作用。
