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Development of anti-inflammatory drugs affecting functions of platelet adhesion molecules

影响血小板粘附分子功能的抗炎药物的开发

基本信息

批准号：
06557134
负责人：
TSUJI Tsutomu
金额：
$ 5.06万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557134/
关键词：
PLATELET LEUKOCYTE CELL ADHESION INFLAMMATION OXYGEN RADICAL CYTOKINE SELECTIN CARBOHYDRATE CHAIN TNF スーパーオキシド

项目摘要

The purpose of this research project was the establishment of the basic concepts of the development of anti-inflammatory drugs affecting the cell adhesion between blood platelets and leukocytes. We got the following results.(1) The screening methods of anti-platelet drugs modifying the functions of platelet adhesion molecules have been developped, where we utilized flow cytometry with fluorescence-labeled platelets and unlabeled leukocytes (2) Recombinant soluble P-selectin inhibited the superoxide anion production in leukocytes stimulated by formyl peptides or tumor necrosis factor-alpha (TNF-alpha). However, the soluble P-selectin had no effect on the oxygen radical production in these cells stimulated by phorbor ester or arachidonic acid. (3) The Neutrophils pretreated with interleukin-8 produced the oxygen radical in response to the soluble P-selectin. (4) When monocytes derived from peripheral blood were incubated in a plate which had been coated with P-selectin, these cells produced TNF-alpha. The optimal concentration of P-selectin for the coating of the plates was found to be 0.3 mug/ml. (5) When leukocytes were incubated with immobilized P-selectin, they peoduced superoxide anion and TNF-alpha. The distribution of P-selectin ligand, sialyl Lewis X carbohydrate, on these cells was examined by immuno-fluorescence microscopy. This carbohydrate epitopes were found to form a cluster at the bottom of the cell, which is the attachment site to the plate. This observation suggested the possible relationship between the leukocyte activation by P-selectin and the cluster formation of its carbohydrate ligand.

本研究项目的目的是建立影响血小板和白细胞之间细胞粘附的抗炎药物开发的基本概念。我们得到了以下结果。(1)利用流式细胞仪对荧光标记血小板和未标记白细胞进行了筛选，建立了改变血小板粘附分子功能的抗血小板药物的筛选方法。（2）重组可溶性P-选择素抑制甲酰基肽或肿瘤坏死因子-α（TNF-α）刺激的白细胞产生超氧阴离子。可溶性P-选择素对佛波酯和花生四烯酸刺激的细胞产生氧自由基无影响。(3)经白细胞介素8预处理的中性粒细胞对可溶性P-选择素产生氧自由基。(4)当来自外周血的单核细胞在用P-选择素包被的平板中孵育时，这些细胞产生TNF-α。发现用于包被板的P-选择素的最佳浓度为0.3 μ g/ml。(5)当白细胞与固定的P-选择素孵育时，它们产生超氧阴离子和TNF-α。用免疫荧光显微镜观察P-选择素配体唾液酸刘易斯X糖在这些细胞上的分布。发现这种碳水化合物表位在细胞底部形成簇，这是与板的附着位点。提示P-选择素对白细胞的激活作用可能与其糖配体簇的形成有关。