【作者】 李肖肖；

【导师】 雷丹青；

【作者基本信息】 广西医科大学 ， 药理学， 2011， 硕士

【摘要】 目的血栓性疾病一直是严重危害人类健康的常见多发性疾病,其发病率、致残率及死亡率都居于高位,它主要危害了心脏、脑、肺等组织器官的血管系统。血栓性疾病的治疗手段主要是溶栓治疗,溶栓药物虽然经历了三代的发展仍然存在一定的副作用。因此,寻找并开发更为安全高效的溶栓药物成为心脑血管性疾病治疗的重要内容。海洋生物因其体内所具有的特殊生理活性物质成为了新药开发的宝贵资源。本实验研究旨在从海洋无脊椎动物方格星虫(Sipunculus Nudus)体内分离纯化出一种新型溶栓物质,并对其主要理化性质及生物活性进行研究。方法(1)采用DEAE Sepharose CL-6B阴离子交换柱层析、Sephadex G-75和Superdex 200凝胶过滤柱层析等多步色谱分离技术相结合分离纯化纤溶酶,用纤维蛋白平板法测定其纤溶活性,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定其相对分子质量。(2)采用纤维蛋白平板法测定其最适温度和最适pH;利用苯甲基磺酰氟(PMSF)、E-64、EDTA、β-ME和抑肽酶5种具有专一性的抑制剂作为其纤溶活性的抑制剂,确定其蛋白酶学分类;通过标准纤维蛋白平板法和加热纤维蛋白平板法,检测其是否具有激酶的活性。(3)采用凝块溶解法测定该酶的体外溶栓效果,溶血性实验,皮下出血性实验和急性毒性实验检测其生物安全性。结果(1)方格星虫纤溶酶在方格星虫体内含量丰富,经过DEAE Sepharose CL-6B,Sephadex G-75和Superdex 200交换层析分离得到纤溶酶,在SDS-PAGE电泳图谱上显示为一条带,其相对分子质量为33.25kDa,纯化倍数为6.48,回收率为10.61%。(2)与蚓激酶作用方式类似,方格星虫纤溶酶具有直接水解纤维蛋白作用;同时通过对纤溶酶原的激活,从而间接水解纤维蛋白,具有激酶活性。(3)酶学相关研究表明:该酶在30-50℃温度范围内,具有优良的热稳定性,其中在40℃时酶活力最高;该酶活力在pH6.5-8.0时较稳定,最适反应pH为7.5;Ba2+离子对该酶活力有一定的抑制作用,其他离子如Mg2+、Ca2+、K+、Na+和Ag+不影响该酶的活性。(4)酶纤溶活性完全被苯甲基磺酰氟(PMSF)抑制,E-64、EDTA、β-ME和抑肽酶对其活性没有影响,是一种典型的丝氨酸蛋白酶。(5)溶血性实验、皮下出血性实验和急性毒性实验证明,该酶具有良好的生物安全性。结论方格星虫纤溶酶具有显著的纤溶活性,且稳定性高,具有潜在的临床研究开发价值。更多还原

【Abstract】 Objective Nowadays, thrombotic disease has become the most severe disease in the world. Thrombotic disease causes not only the high incidence but also high mortality and disability. It mainly damages the blood systems of heart, brain and lung. Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. There are three generations of thrombolytic agents, but more or less side effects appear in each of them. It is important for medicinal experts to search for more safe and effective medicines to cure the patients. In the light of the unique advantage of marine lives, they are becoming more and more precious resources in the future. Sipunculus Nudus is a marine invertebrate animal living in the sediment of South China Sea. It is found that the coelomic fluid of Sipunculus Nudus dose not congeal after being separated for a long time. This suggests that it may contain anticoagulants or fibrinolytic substances. In this study, we try to purify fibrinolytic enzyme from a marine invertebrate-Sipunculus Nudus. In the sametime, we will study its thrombolytic effect and its safety. Methods We used sephacryl gel filtration chromatography and ion exchange chromatography to separate and purify Sipunculus Nudus fibrinolytic enzyme. The purity and relative molecular weight of Sipunculus Nudus fibrinolytic enzyme were identified by SDS-PAGE. The fibrinolytic activity was determined by fibrin plate. Rabbits were used to measure thrombolytic effects.Results (1) The fibrinolytic enzyme could be isolated and purified by using of DEAE Sepharose CL-6B, Sephadex G-75 and Superdex 200 respectively. SDS-PAGE showed that only a single band for the purified enzyme, and the molecular weight was estimated to be about 33.25 kDa.The recovery yield was 10.61%, and the specific activity increased by 6.48 folds over the extracted crude. (2) Sipunculus Nudus fibrinolytic enzyme could not only directly degrade fibrin but also indirectly hydrolyze fibrin through transforming plasminogen into plasmin. (3) The enzyme activity of Sipunculus Nudus fibrinolytic enzyme was stable in the temperature between 30℃and 50℃and the pH ranging from 6.5 to 8.0. The optimum temperature of enzyme was about 40℃and the optimum pH was 7.5 respectively. The enzyme activity was obviously inhibited by Ba2+, other metal ions including Mg2+、Ca2+、K+、Na+、Ag+ had no obvious effect on the enzyme activity. (4) PMSF was inhibitor of Sipunculus Nudus fibrinolytic enzyme. (5) The haemolytic test, hypodermic bleeding test and acute toxicity test were confirmed that Sipunculus Nudus fibrinolytic enzyme owned good bio-security.Conclution Sipunculus Nudus fibrinolytic enzyme could obviously enhance fibrinolytic activity in these experiments. It was safe and effective modern antithrombotic pharmaceutics with potential clinical applied values.更多还原