【作者】 朱世权；

【导师】 许云禄；

【作者基本信息】 福建医科大学 ， 药理学， 2009， 硕士

【摘要】 血小板是血液中最小的、具有多种功能的细胞,它在血栓形成、动脉粥样硬化等病理过程中扮演了重要的角色。蛇毒中广泛存在抑制血小板聚集的活性组分,它们的生化特性及作用机理均不尽相同。近年来,已有文献报道了从不同的种属的蛇毒,特别是从蝰科蝮亚科毒蛇所分泌的蛇毒中提取及分离出多种血小板聚集抑制剂,并对它们的活性进行了分析。关于尖吻蝮蛇毒这方面的研究较少,本课题拟从尖吻蝮蛇蛇毒(Agkistrodon acutus venom,AAV)中分离纯化出具有的抗血小板聚集作用组分,测定其理化性质,观察其体内外抗血小板聚集活性,为进一步研究开发提供实验依据。1.抗血小板聚集组分的分离纯化应用Superdex 75凝胶过滤色谱,从尖吻蝮蛇粗毒中分离得到6个蛋白峰;其中蛋白峰Ⅳ抑制二磷酸腺苷(ADP)诱导的血小板聚集活性最强,该组分再经Sephadex G-25凝胶色谱、DEAE Sepharose Fast Flow离子交换色谱和Lichrospher C18反相色谱等纯化得一个电泳纯蛋白组分,暂定名为AAV-IV3-2。SDS—PAGE蛋白电泳鉴定发现,AAV-IV3-2呈单一条蛋白区带。2. AAV-IV3-2部分理化性质测定根据AAV-IV3-2和Marker在凝胶上的相对位置,利用Image Master VDS凝胶成像系统中的软件计算得出AAV-IV3-2的分子量大约为15.3kDa。3.AAV-IV3-2对血小板聚集的影响采用经典的Born法,发现AAV-IV3-2在体外可抑制ADP和凝血酶诱导的血小板聚集,且该作用呈明显的量效关系,AAV-IV3-2对ADP、凝血酶诱导的血小板聚集的IC50分别为1.78μg/ml和5.06μg/ml。4、AAV-IV3-2对ADP-Na诱导的小鼠急性肺栓塞的影响采用ADP-Na诱导的小鼠急性肺栓塞模型发现, AAV-IV3-2 iv可以明显缩短小鼠呼吸窘迫时间,中高剂量组(150、200μg/kg)小鼠恢复正常呼吸、自主活动的时间(秒)分别为114.2±31.53和101.9±42.19,与生理盐水组(176.6±57.52)相比均有明显缩短(P<0.05)。提示AAV-IV3-2在体内具有抗血小板作用。结论:应用凝胶过滤和离子交换色谱方法可以从尖吻蝮蛇毒中分离得到一个相对分子量为15.3kDa、纯度为电泳纯的蛋白组分AAV-IV3-2。AAV-IV3-2能抑制ADP和凝血酶诱导的血小板聚集,并有明显的量效依赖关系;在小鼠体内也具有抗血小板作用。更多还原

【Abstract】 Snake venom was known to possess components affecting platelet function in various ways. Screening anti-platelet aggregation protein from snake venom becomes an intriguing work because of their potent clinical use in treating abnormal platelet activation and arterial thrombus. This study was designed to isolate and purify anti-platelet aggregation protein from Agkistrodon acutus venom(AAV) and analyse its characterization and biological activities.1. Purification and characterization of the anti-platelet aggregation protein from Agkistrodon acutus VenomSix fractions(Ⅰ～Ⅵ) were isolated from AAV by Gel filtration chromatography on Superdex 75 .The fractionⅣpossess the best anti-platelet aggregation activity, so it suggested as a contained the anti-platelet aggregation component. An anti-platelet aggregation protein named AAV-IV3-2 was purified from the fraction by sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was homogeneous as a single band showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE) with molecular weight 15.3kDa as calculated by Image Master VDS system.2. The effects of AAV-IV3-2 on the human platelet aggregation.According to the Born’s method, the effects of AAV-IV3-2 on platelet aggregation induced by ADP and thrombin were assayed. The results showed that AAV-IV3-2 dose-dependently inhibited ADP- and thrombin-induced platelet aggregation with IC50 1.78 and 5.06μg/ml respectively.3. The effect of AAV-IV3-2 on the time of respiratory distress of acute pulmonary thrombosis in mice induced by ADP-Na. The time of respiratory distress of acute pulmonary thrombosis in mice of middle, high dose groups(114.2±31.53、101.9±42.19,S)respectively were remarkably shorter than that of NS group (176.6±57.52,S)(P<0.05).CONCLUSIONAAV-IV3-2 was purified from Agkistrodon acutus venom with molecular weight 15.3kDa. It was a potent platelet aggregation inhibitor.更多还原