Role of leukocyte-endothelial interaction for pathogenesis of vessel diseases perturbed by cytokines

为探讨白细胞-内皮细胞相互作用在细胞因子干扰的血管疾病发病机制中的作用，进行了体外实验。血流动力调节内皮细胞的各种功能，即使在基因调控下存在细胞因子的情况下也是如此。用改进的锥板式粘度计研究了剪切力对培养的人脐静脉内皮细胞(HUVECs)在细胞因子或脂多糖(LPS)扰动下凝血和纤溶系统的影响。目前已研制出几种抗人鼻腔粘膜微血管的单抗。针对炎症部位下白细胞与内皮细胞黏附的重要分子--E-选择素和Mel-CAM的两种黏附糖蛋白，已研制出抗E-选择素和Mel-CAM的单抗。静息状态下未刺激的人脐静脉内皮细胞未检测到E-选择素的表达，而其表面膜上E-选择素的…表达增加肿瘤坏死因子刺激后3~6h更明显。而静息状态下未刺激的HUVECs明显表达MEL-CAM，而培养上清液中MEL-CAM的释放量在肿瘤坏死因子刺激后增加，在剪切力作用下减少。免疫印迹法检测ICAM-1和VCAM-1在人脐静脉内皮细胞中的表达。静息状态下未刺激的HUVECs中未检测到ICAM-1和VCAM-1的表达，而在肿瘤坏死因子刺激后3~6h，ICAM-1和VCAM-1的表达增强。我们用Ficoll梯度法从人外周血中分离出单个核细胞。未受刺激的HUVECs对MNCs的黏附作用不明显，而在肿瘤坏死因子刺激后6~9h，MNCs与HUVECs的黏附明显。有趣的是，这些细胞黏附分子的表达和白细胞与肿瘤坏死因子刺激的人脐静脉内皮细胞的黏附已经通过抑制NF-κB的激活而减少。这些结果表明，在肿瘤坏死因子刺激下，通过激活NF-κB来调节白细胞与内皮细胞的相互作用，可能是通过细胞黏附分子调节炎症和动脉粥样硬化的重要因素。较少

To investigate the role of leukocyte-endothelial interaction for pathogenesis of vessel diseases perturbed by cytokines, in vitro experiments have been undertaken. Hemodynamic forces modulate various endothelial cell functions even in the presence of cytokines under gene regulation. We have investigated the effect of shear stress on coagulation and fibrinolysis system in cultured human umbilical vein endothelial cells (HUVECs) perturbed by cytokines or lipopolysaccharide (LPS), using modified cone-plate type viscometer, in which well controlled and defined shear forces were generated. Several monoclonal antibodies have been developed against human nasal-mucous microvessels. Monoclonal antibodies have been produced against two adhesive glycoproteins for Eselectin and Mel-CAM that act as important molecules for leucocyte adhesion to endothelium under inflammatory site. E-selectin was not detected in resting unstimulated HUVECs, whereas, surface membrane expression of E-selectin was incre … More ased 3〜6 hours after the stimulation of TNF. In contrast, Mel-CAM expression was clearly observed in resting unstimulated HUVECs, whereas the release to culture supernatant was increased after TNF stimulation and decreased under shear stress. We also investigated the expression of ICAM-1 and VCAM-1 in HUVECs, using western blotting. ICAM-1 and VCAM-1 was not detected in resting unstimulated HUVECs, whereas, the expression of ICAM-1 and VCAM-1 have been increased 3〜6 hours after the stimulation of TNF. We have isolated mononuclear cells (MNCs) from human peripheral blood, using Ficoll-gradient methods. No adhesion of MNCs was observed to resting unstimulated HUVECs, whereas, the leukocyte adhesion to HUVECs was clearly observed 6〜9 hours after TNF-stimulation. Interestingly the expression of these cytoadhesion molecules and leukocyte adhesion to TNF-stimulated HUVECs have been decreased by the inhibition of NF-κB activation. These results indicate that the regulation of leukocyte-endothelial interaction under TNF-stimulation through NF-κB activation might be very important modulator for inflammation and atheroclerosis through cytoadhesion molecules. Less