榆干离褶伞溶栓酶的溶栓作用及血管内皮细胞的保护作用研究

Thrombosis is a kind of cardiovascular diseases that endanger human health, and the incidence of the desease is increasing year by year and younger on average. At present, the fibrinolytic agents available for clinical use have some side effects, such as bleeding and fever, however. So, it becomes hot spot to find an effective fibrinolytic agent of natural source. In this study we will select fibrinolytic enzyme from Lyopyllum ulmarium of Changbai mountain as the object, and study the thrombolytic effect of the fibrinolytic enzyme from Lyopyllum ulmarium and its effects on functions of the coagulation and fibrinolysis system in animal models of thrombosis. Because bioactivators from vascular endothelial cells have a critical function in the regulation of vascular tone, antiplatelet aggregation, and anticoagulant fibrinolysis, thrombosis is closely related to the injuries of vascular endothelial cells. In this project, we will establish the vascular endothelial cell injury model, study the effect of fibrinolytic enzyme from Lyopyllum ulmarium on proliferation, cell cycle, apoptosis and apoptosis-related protein expression, and the secretion fuction of vaso-active substances in vascular endothelial cells, and investigate its antithrombotic and thrombolytic mechanisms from different angles, such as anticoagulant fibrinolytic, antioxidative, and vascular endothelial cell protective activities. This project will provide an experimental basis for development of new thrombolytic agents and exploitation of our rich mushroom resources, and has a very important application value .

血栓症是严重危害人类健康的心血管疾病之一，其发病率逐年上升且趋于年轻化。目前临床上所用的溶栓药物具有出血、发热等诸多不良反应，因此开发天然来源的新型有效溶栓药物成为研究的热点。本项目以长白山野生榆干离褶伞中提取的溶栓酶作为研究对象，通过建立血栓动物模型，研究榆干离褶伞溶栓酶的溶栓作用和它对凝血及纤溶系统功能的影响。因血管内皮细胞分泌的生物活性物质在调节血管紧张度、抗血小板聚集以及抗凝促纤溶等过程中起重要作用，所以血管内皮细胞的损伤与血栓形成密切相关。本项目中通过建立血管内皮细胞损伤模型，探讨榆干离褶伞溶栓酶对血管内皮细胞的增殖、细胞周期、凋亡及凋亡通路中相关蛋白的表达和血管活性物质分泌功能的影响，从抗凝促纤溶、抗氧化以及保护血管内皮细胞等不同角度探讨其溶栓机制。此项研究对新型的溶栓药物的开发以及我国丰富的食用菌资源的开发提供实验依据，具有十分重要的应用价值。

血栓症严重危害人类健康的心血管疾病，其致残率和死亡率逐年上升，且趋于年轻化。因此，对血栓性疾病的预防及治疗越来越受到人们的关注。前期研究中我们发现榆干离褶伞在体外具有溶栓作用。因此，在本研究中我们主要探讨了其菌丝体中分离纯化的榆干离褶伞溶栓酶（Lyophyllum Ulamarium fibrinolytic enzyme, LUFE）的抗栓作用和其机制。.本课题通过建立大鼠血栓模型、血淤模型和前弥漫性血管内凝血模型，探讨了LUFE的抗栓作用，并从抗氧化、抗炎、抑制血小板活化、调节凝血纤溶功能等多角度阐明了其抗栓机制。在体外，以过氧化氢、乙醇、脂多糖等诱导血管内皮细胞损伤模型，观察了LUFE对其结构及功能的影响。体内实验研究结果表明，LUFE抑制血栓的形成、减轻血管内膜的损伤、调节血管内皮细胞的分泌功能并抑制血小板的活化、同时提高机体的抗氧化水平，对受损的肝、肾、肺等组织有保护作用。细胞实验结果表明，LUFE提高血管内皮细胞的存活率、抑制细胞内活性氧的生成、提高线粒体跨膜电位并抑制细胞凋亡。从分子水平上，LUFE抑制线粒体凋亡途径中相关蛋白的表达、并通过对TLR4/ TAK1/NF-κB/COX2通路、NF-κB - iNOS - NO通路的调控作用来对血管内皮细胞起保护作用。通过本课题的研究已明确了榆干离褶伞的抗栓作用、抗氧化、抗炎作用及对血管内皮细胞的保护作用，为榆干离褶伞的药理作用提供实验依据，也为新型的抗栓药物的开发以及我国丰富的食用菌资源的开发提供实验依据，具有十分重要的应用价值。