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Evaluation of antioxidant activity against atherosclerosis using co-culture system

使用共培养系统评价抗动脉粥样硬化的抗氧化活性

基本信息

批准号：
11672208
负责人：
NOGUCHI Noriko
金额：
$ 2.56万
依托单位：
Research Center for Advanced Science and Technology, University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672208/
关键词：
Co-culture Atherosclerosis Oxidation LDL Antioxidants Cytokine

项目摘要

To analyze in vitro the migration of monocytes to the subendothelial space, their differentiation into macrophages, and the subsequent formation of form cells in vitro, we developed a 2-coculture system with rabbit aortic endothelial cells, aortic smooth muscle cells, and a mixture of matrix proteins on polyethylene folters in chemotaxis chambers. When oxidized low density lipoprotein (LDL) was added to the matrixlayer of coculture, on day 4 transmigrant cells exhibited lipid deposit droplets, and by day 7, they had the appearance of typical foam cells.To follow lipid oxidation of low density lipoprotein andwithin cells we developed a novel method using diphenyl-1-pyrenylphosphine (DPPP) as a fluorescent probe. DPPP reacted with organic hydroperoxides and hydrogen peroxide stoichiometrically to give DPPP oxide (DPPP=O). DPPP incorporated into phosphatidylcholine liposomal membranes and polymorphonuclear leukocytes (PMNs) reacted with methyl linoleate hydroperoxide rapidly but not with … More hydrogen peroxide nor with tert-butyl hydroperoxide. This novel method revealed that lipid peroxidation proceeded within membranes of PMN stimulated by phorbol-12-myristate-13-acetate, which is known to produce several kinds of free radicals. It was found that DPPP is a suitable fluorescent probe to monitor lipid peroxidation within the cell membranes specifically. DPPP incorporated into LDL gave a strong fluo-rescence within macrophage after macrophage took up DPPP-containing LDL.We examined antioxidant activity of 4 isomers of vitamin E, (α- and γ-) tocopherol and tocotrienol, and inhibitory effect on expression of adhesion molecule of human endothelial cell. There were not significant deffereces in antioxidant activity and inhibitory effect on adhesion molecule expression. However, the concentrations of tocotrienols within cell were more than 10 times higher than that of tocopherol due to double bond in side chain of tocotrienol. By taking the defference in the concentrations of these antioxidants into considerations , tocopherols were more efficient in inhibition of the expression of adhesion molecules than tocotrienols. It is interesting to investigate these effects in coculture system we developed. Less

为了分析在体外单核细胞的迁移到内皮下的空间，他们分化成巨噬细胞，并随后形成的形式细胞在体外，我们开发了一个2-共培养系统与兔主动脉内皮细胞，主动脉平滑肌细胞，和混合物的基质蛋白聚乙烯folters在趋化性室。在共培养的基质层中加入氧化低密度脂蛋白（LDL），第4天移行细胞出现脂存款滴，第7天移行细胞出现典型的泡沫细胞。DPPP与有机过氧化氢和过氧化氢按化学计量反应，得到DPPP氧化物（DPPP=O）。DPPP掺入磷脂酰胆碱脂质体膜和多形核白细胞（PMNs）与亚油酸甲酯过氧化氢迅速反应，但不与亚油酸甲酯过氧化氢反应。 ...更多信息过氧化氢也不与叔丁基过氧化氢反应。这种新的方法表明，脂质过氧化反应的中性粒细胞膜内的佛波醇-12-肉豆蔻酸酯-13-乙酸酯刺激，这是已知的产生几种自由基。结果表明，DPPP是一种特异性监测细胞膜脂质过氧化反应的荧光探针。巨噬细胞摄取含DPPP的LDL后，DPPP掺入LDL，在巨噬细胞内产生强荧光，我们检测了维生素E的4种异构体（α-和γ-）生育酚和生育三烯酚的抗氧化活性，以及对人内皮细胞粘附分子表达的抑制作用。抗氧化活性和对粘附分子表达的抑制作用无显著差异。然而，由于生育三烯酚侧链中的双键，生育三烯酚在细胞内的浓度比生育酚高10倍以上。通过考虑这些抗氧化剂浓度的差异，生育酚比生育三烯酚更有效地抑制粘附分子的表达。在我们开发的共培养系统中研究这些效应是有趣的。少