|Budget Amount *help
¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1999 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1998 : ¥1,500,000 (Direct Cost : ¥1,500,000)
(1) We have developed a new device which can sensitively detect the presence of platelet aggregates and platelet-neutrophil aggregates in whole blood. A pulse laser and a CCD camera are attached to a flow cytometer, which is an automated reticulocyte analyzer, R-3000 (Sysmex, Japan). Forward scatter intensity and fluorescence intensity of auramine O, a dye for staining RNA and DNA, are used to selectively gate platelet aggregates and platelet-neutrophil aggregates. Cells that fall in the gated area are checked for their images to correctly distinguish these aggregates from other cell components.
Upon stimulation with ADP or serotonin, formation of platelet-neutrophil aggregates was clearly detected with this new device. Now we are trying to develop a system which enables its quantitative measurement.
(2) We examined the sphingolipid metabolism of peripheral blood cells, i.e., platelets, erythrocytes, neutrophils, and mononuclear cells. A distinguishing characteristic of sphingolipid meta
bolism in these highly-differentiated cells was their high sphingosine (Sph) kinase activity. About 40% of platelet Sph 1-phosphate (Sph-1-P) could be released extracellularly by 12-O-tetradecanoylphorbol 13-acetate, possibly through mediation by protein kinase C. On the other hand, in erythrocytes, neutrophils, and mononuclear cells, a significant percentage of Sph-1-P formed inside the cell was discharged without stimulation, while the stimulation-dependent release was marginal. Sph and its methylated derivative, N,N-dimethylsphingosine, induced apoptosis not only in neutrophils but also in mononuclear cells, while Sph-l-P elicited CaィイD22+ィエD2 mobilization in platelets. Our results suggest that all blood cells may remove plasma Sph, which is harmful or suppressive to cellular functions, and change it into Sph-1-P, acting as the source of plasma Sph-1-P, which may play a variety of important roles in blood vessels.
我々は,活性化血小板からsphingosine 1-phosphate(Sph-1-P)が放出されること,スフィンゴ脂質の観点より血小板―各種血球・血管内皮細胞クロストークを考えることの重要性を報告してきた.好中球に対して,Sph-1-Pは明らかな作用を示さなかったが,ジ メチルスフィンゴ シン・Sph・セラミドは,この順で,好中球アポトーシスを誘導することを形態学的変化・フローサイトメトリー法にて確認した.血管内皮細胞では,Sph-1-P産生に対するSphキナーゼ阻害剤の効果の検討を行い,Sph-1-Pが血小板内セカンド・メッセンジャーとして作用しえないことを明らかにした. Less