Study on mechanism of thrombosis and hemostasis in endothelium

为了探讨内皮细胞血栓形成和止血的调控机制，我们进行了体外实验。血流动力学力调节各种内皮细胞功能，甚至在基因调控下存在细胞因子。我们研究了剪切应力对受细胞因子或脂多糖（LPS）干扰的培养人脐静脉内皮细胞（HUVECs）凝血和纤溶系统的影响，使用改进的锥形板型粘度计，在该粘度计中产生良好控制和定义的剪切力。我们发现细胞因子诱导的组织因子表达在流动的反应中降低，而组织纤溶酶原激活剂的分泌在细胞因子存在的剪切应力下增加，纤溶酶原激活剂抑制剂-i的分泌在细胞因子存在的情况下增加，但在剪切应力下减少。此外，我们还利用免疫细胞化学、EIA法和激光流式细胞术研究了剪切胁迫下HUVECs中其他因子的表达。组织因子通路抑制剂（Tissue factor pathway inhibitor， TFPI），作为组织因子的调节因子的丝氨酸蛋白酶，不受炎症细胞因子TNF和剪切应力的刺激影响，而受促瘤激活剂PMA的抑制。接下来，开发了几种针对人鼻粘膜微血管的单克隆抗体。针对炎性部位白细胞粘附内皮的重要分子e -选择素和Mel-CAM两种黏附糖蛋白已制备出单克隆抗体。静止未受刺激的HUVECs中未检测到e -选择素，而TNF刺激后3-6小时，e -选择素的表面膜表达增加。相比之下，Mel-CAM在静息未受刺激的HUVECs中明显表达，而TNF刺激后向培养上清的释放增加，剪切应力下减少。提示血栓形成和止血的机制可能受到复杂网络的调控。剪切力和细胞因子调控机制的交叉对话，至少作为一部分，是血栓形成和抗血栓形成的重要调节剂。少

To investigate the regulatory mechanism of thrombosis and hemostasis in endothelium, 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. We have revealed that cytokine-induced tissue factor expression was decreased in response to flow, whereas secretion of tissue plasminogen activator was augmented under shear stress in the presence of cytokines, and the secretion of plasminogen activator inhibitor-I was increased in the presence of cytokines, but, decreased under shear stress. Furthermore, we investigated the expression of various other factors in HUVECs under shear stress … More in the presence of cytokines, using immunocytochemistry, EIA method, and laser flow-cytometry. Tissue factor pathway inhibitor (TFPI), serine protease that acts as a regulator for tissue factor, was not affected by stimulation of inflammatory cytokines, TNF, nor shear stress, but decreased by tumor promoting activator, PMA. Next, several monoclonal antibodies have been developed against human nasal-mucous microvessels. Monoclonal antibodies have been produced against two adhesive glycoproteins for E-selectin 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 increased 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. These results indicate that the mechanism of thrombosis and hemostasis might be regulated by complicated net-work. The cross-talk of regulatory mechanism for shear forces and cytokines, at least as a part, is important modulator for thrombogenecity and anti-thrombogenecity. Less