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

为了在体外分析单核细胞向亚皮细胞的迁移，它们的分化为巨噬细胞以及随后在体外形成细胞的形成，我们开发了一种2个培养系统，该系统与兔主动脉内皮细胞，主动脉平滑肌细胞以及基质蛋白在化学素叶片中的混合物中的化学蛋白质的混合物形成了化学素质素的化学素叶片。当将氧化的低密度脂蛋白（LDL）添加到共培养基质剂时，在第4天，在第4天传播脂质沉积液滴时，到第7天，它们出现了典型的泡沫细胞。探测。 DPPP与有机氢过氧化物和过氧化氢反应，从而得到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）中，与甲基linoleate氢过氧化物反应迅速，但与…更多的过氧化氢或与Tert叔丁基水氧化物反应。这种新颖的方法表明，脂质过氧化是在phorbol-12-摩尔斯酯-13-乙酸盐刺激的PMN机制中进行的，该机制已知会产生几种自由基。发现DPPP是一种合适的荧光探针，可特别监测细胞膜内的脂质过氧化。巨噬细胞摄入含DPPP的LDL后，掺入LDL中的DPPP在巨噬细胞内产生了强烈的荧光般的敏感。我们检查了4种维生素E（α-和γ-）生育酚和生育酚的抗氧化剂活性，以及对人内皮细胞表达的抑制作用。抗氧化活性和抑制作用对粘合分子表达没有明显的脱育。然而，由于在生育三烯醇的侧链中，由于细胞内的生育三烯醇的浓度比生育酚高10倍以上。通过考虑这些抗氧化剂的浓度下降，生育酚在抑制粘合分子表达的效率比植素醇更有效。在我们开发的共培养系统中研究这些影响很有趣。较少的