Evaluation of antioxidant activity against atherosclerosis using co-culture system

为了在体外分析单核细胞向内皮下间隙的迁移、向巨噬细胞的分化以及随后在体外形成形细胞的过程，我们在趋化小室中建立了兔主动脉内皮细胞、主动脉平滑肌细胞和基质蛋白混合物的2-共培养体系。将氧化低密度脂蛋白(LDL)加入到共培养基质层中，移植细胞在第4天出现脂滴，第7天出现典型的泡沫细胞，为了跟踪低密度脂蛋白在细胞内和细胞内的脂质氧化，我们开发了一种新的方法，使用二苯基-1-丙二烯基膦(DPPP)作为荧光探针。DPPP与有机过氧化氢和过氧化氢按化学计量比反应生成DPPP氧化物(DPPP=O)。磷脂酰胆碱脂质体膜和多形核白细胞(PMN)与亚油酸甲酯过氧化氢反应迅速，但不与…反应更多的过氧化氢和叔丁基氢过氧化氢。这一新的方法揭示了佛波醇-12-肉豆蔻酸酯-13-乙酸酯刺激的PMN膜内发生的脂质过氧化，已知产生了几种自由基。研究发现，DPPP是一种适合于特异性监测细胞膜内脂质过氧化的荧光探针。研究了维生素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，以及对人内皮细胞黏附分子表达的抑制作用。在抗氧化活性和对黏附分子表达的抑制作用方面无明显差异。然而，由于生育三烯酚侧链上的双键，细胞内生育三烯醇的浓度是生育酚的10倍以上。考虑到这些抗氧化剂浓度的差异，生育酚比生育三烯醇更有效地抑制黏附分子的表达。在我们开发的共培养系统中研究这些影响是很有趣的。较少

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