Yang Limin, Zhu Xuewei, Zhao Xia, Deng Zhongduan
Department of Pathology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030.
J Huazhong Univ Sci Technolog Med Sci. 2003;23(3):219-22, 233. doi: 10.1007/BF02829496.
In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
为研究二酰胺诱导发生脂质过氧化的内皮细胞（ECs）是否能表达和分泌巨噬细胞炎性蛋白1α（MIP-1α），将ECs暴露于不同浓度的二酰胺4小时后，采用细胞酶联免疫吸附测定（ELISA）检测细胞中MIP-1α蛋白的表达，并用细胞原位杂交和核酸酶S1保护试验测定MIP-1α mRNA的表达。采用改良的Boyden小室微孔滤膜法检测MIP-1α的趋化活性。细胞ELISA显示，暴露于1μmol/L、5μmol/L和10μmol/L二酰胺的内皮细胞中MIP-1α蛋白的表达分别是对照细胞的1.9倍、2.3倍和1.7倍，经方差分析具有统计学意义。原位杂交显示，用1μmol/L、5μmol/L和10μmol/L二酰胺处理的ECs的mRNA表达分别是对照组的1.3倍、3.0倍和1.7倍，具有统计学意义（F = 188.93，P < 0.01）。用核酸酶S1保护试验测定，5μmol/L二酰胺处理的ECs的mRNA表达是对照组的3.4倍（t = 8.70，P < 0.05）。5μmol/L二酰胺处理的ECs条件培养基的趋化反应（99.50±4.31μm）强于ECs条件培养基的趋化反应（66.47±3.25μm）（F = 404.31，P < 0.05），加入MIP-1α抗体后趋化反应显著降低（F = 192.25，P < 0.05）。这表明脂质过氧化诱导剂二酰胺可刺激ECs产生高水平的MIP-1α，并可能通过促进外周血单核细胞向动脉内膜迁移在动脉粥样硬化形成中起重要作用。
