缺氧-再氧化条件下血小板对白细胞-肝窦内皮细胞黏附及跨内皮迁移的影响

Effects of platelets on the adhesion between leukocytes and liver sinusoidal endothelium cells as well as on the transendothelial migration of leukocytes undergoing hypoxia-reoxygenation

目的分析血小板在缺氧-再氧化条件下对白细胞-肝窦内皮细胞间黏附和跨内皮迁移的影响及相关机制。方法人肝窦内皮细胞（LSEC）经缺氧24h、复氧2h（HR）模拟肝脏缺血-再灌注（IRI）过程。将血小板和白细胞用荧光染料标记，通过荧光显微镜、连续光谱荧光仪及激光共聚焦显微镜检测细胞黏附和迁移情况；通过抗体阻断实验分析相关细胞黏附分子。结果血小板在HR的LSEC上黏附明显增加，黏附的血小板的荧光强度为289．17±20．00，与正常培养组（142．10±7．53）相比差异有统计学意义（P〈0．01）。HR＋PLT组中黏附的血小板明显促进白细胞在HR的LSEC上的黏附，与HR组相比差异有统计学意义（360．71±23．47比186．39±17．96，P〈0．01），这种白细胞黏附分别被抗黏附分子糖蛋白Ⅰb（GPⅠb）、糖蛋白Ⅱb（GPⅡb）、糖蛋白Ⅲa（GPⅢa）、P-选择素（P-selectin）、CD31、细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）、内皮细胞白细胞黏附分子-1（ELAM-1）的抗体阻断。激光共聚焦显微镜显示，血小板位于白细胞与LSEC之间，介导白细胞与LSEC的黏附。HR＋PTJT组添加的血小板抑制白细胞的跨内皮迁移，迁移白细胞的荧光强度为167．27±10．92，与HR组（227．79±16．51）相比差异有统计学意义（P〈0．05）。结论缺氧-再氧化条件下，血小板黏附在肝窦内皮细胞表面，增加白细胞-内皮细胞的黏附，并抑制白细胞的跨内皮迁移，从而导致白细胞在肝脏血管内的扣押。

Objective To investigate the effects of platelet on intercellular adhesion between leukocyte and liver sinusoidal endothelial cell（LSEC） and the transendothelial migration under the hypoxia- reoxygenation condition, as well as the role of relevant adhesion molecules. Method LSEC was cultured for 24 hours under hypoxia condition and then reoxygenated for 2 hours （ hypoxia-reoxygenation, HR）. This hypoxia-reoxygenation model was used to simulate the clinical liver ischemia-reperfusion injury process （IRI）. Platelets and leukocytes were labeled with fluorescence dye, and then the adhesion was detected by fluorescence microscope, fluorescence plate reader and laser scanning confocal microscope. Antibody blockage experiment was used to analyze the relevant adhesion molecules. Results The adhesion between platelets and LSEC was increased significantly after HR. The fluorescence intensity of adherent platelets increased from 142. 10 ± 7. 53 to 289. 17± 20. 00 （ P 〈 0. 01 ）. After H-R treatment and the addition of platelets, the number of adherent leukocytes increased markedly, and a significant statistical difference （ 360. 71 ± 23.47 and 186. 39 ± 17.96, P 〈 0. 01 ） was found in comparing with the platelet deficient group. These adhesion processes could be blocked respectively by anti-GPⅠ b, anti-GP Ⅱ b, anti-GP Ⅲ a, anti-P- selectin,anti-CD31 ,anti-ICAM-1 ,anti-VCAM-1 and anti-ELAM-1. Confocal microscopy showed that platlets located between leukocytes and LSEC, and mediated their adhesion process. However, the adhesion of platelets to LSEC decreased the transendothelial migration of leukocytes （227.79 ± 16. 51 and 167.27 ± 10. 92, P 〈 0.05 ）. Conclusion During ischemia-reperfusion condition platelets adhere to the surface of LSEC, and then further mediate more adhesion processes between leukocytes and endothelial cells, as well as inhibit the transendothelial migration of leukocytes. The consequence is that large numbers of lenkocytes were sequestrated within hepatic sinus, and deteriorate liver ischemia-reperfusion iniury.