皖南蝮蛇毒血小板抑制因子（AHV-PI）的理化特性与急性毒性研究

发布时间：2018-06-16 14:59

  本文选题：蛇毒 + AHV-PI　； 参考：《皖南医学院》2017年硕士论文

【摘要】：目的:观察(AHV-PI)对小鼠的急性毒性作用,探讨该组分相关的理化性质。方法:1.采用阴阳离子交换法和分子筛蛇毒蛋白纯化柱纯化皖南蝮蛇粗毒,纯化后通过抑制血小板聚集来筛选目的峰即为AHV-PI,将目的峰冻干备用。2.小鼠急性毒性实验:半数致死量法,将小鼠分为空白对照组,AHV-PI1-AHV-PI5组,空白对照组为生理盐水溶液,AHV-PI组分别加入9.0、10.7、12.7、15.1、18.0mg/kg的AHV-PI溶液,将小鼠动物体温计塞入小鼠肛门内待小鼠平稳后测量小鼠基础体温,然后腹腔注射配制好的溶液,观察并记录小鼠精神状况和体温变化,死亡小鼠记录死亡时间后立即取出小鼠心肝肺等全身脏器用10%甲醛(福尔马林)固定液固定。未死亡小鼠分别测定体温,观察一周后全部眼球取血测量血粘度变化,然后处死存活小鼠并解剖,取下各个脏器行HE染色,3.蛋白等电点和分子量测定:双向电泳第一项等点聚焦,然后配胶行第二项垂直电泳,脱色后凝胶成像仪拍照分析AHV-PI等电点和分子量。结果:1.皖南蝮蛇毒毒素之中包含有AHV-PI组分,经过阴阳离子交换以及分子筛以后能够分离出体外强烈抑制血小板的该组分。2.皖南蝮蛇毒血小板抑制因子小鼠急性毒性作用可知,小鼠腹腔注射AHV-PI的LD50为13.44mg/kg其95%可信限为(12.72~14.16)mg/kg。3.AHV-PI腹腔注射小鼠体内后小鼠体温会出现短暂性下降2~5℃不等,在一定程度上随着AHV-PI浓度的升高,体温降低的越明显,约2小时后未死亡小鼠体温逐渐恢复正常。4.双向电泳结果显示蛇毒纯化后组分AHV-PI经过双向电泳后可见分子量几乎相同的五组电荷相差较小的蛋白质点。拍照软件分析后可得其相对分子量约为34.2KD,等电点约为4.94~4.98之间。结论:1.AHV-PI是一种小分子,偏酸性的蛇毒组分蛋白质。2.根据前期该组分药理学相关研究,有效作用范围ED50在0.1mg/kg左右,TI=LD50/ED50,比值相差一百多倍,属于使用范围较为安全的活性组分。3.AHV-PI可以引起血粘度降低,机体凝血功能障碍,导致出血倾向加剧,其中肺组织出血是最为严重的毒副反应。4.在蛇毒天然毒素毒性研究中存活动物中毒后的的体温变化是相对有价值的预测因子。可以作为未来蛇毒活性组分用药安全重要的监测指标。
[Abstract]:Aim: to observe the acute toxicity of AHV-PI to mice and to explore the physicochemical properties of its components. Method 1: 1. The crude venom of Agkistrodon halys was purified by anion exchange method and molecular sieve snake venom protein purification column. After purification, the target peak AHV-PIwas screened by inhibiting platelet aggregation, and the target peak was freeze-dried. Acute toxicity test: the mice were divided into control group (AHV-PI1-AHV-PI5) by 50% lethal dose method. The blank control group was treated with normal saline solution AHV-PI (9.0mg / kg AHV-PI). The animal thermometer was inserted into the anus of the mice and the basic body temperature of the mice was measured after the mice were stabilized. Then the mice were injected intraperitoneally with the prepared solution to observe and record the changes of the mental state and body temperature of the mice. The dead mice were fixed with 10% formaldehyde (formalin) immediately after recording the time of death. The body temperature of the non-dead mice was measured and the blood viscosity was measured after a week's observation. Then the surviving mice were killed and dissected and the organs were taken out for HE staining. Protein isoelectric point and molecular weight measurement: the first isoelectric point focus of two-dimensional electrophoresis and the second vertical gel electrophoresis were used to analyze AHV-PI isoelectric point and molecular weight after decolorization by gel imager. The result is 1: 1. Agkistrodon halys venom contains AHV-PI component. After anion exchange and molecular sieve, we can isolate the component of AHV-PI, which can strongly inhibit platelets in vitro. The acute toxicity of platelet inhibitor of Agkistrodon halys venom showed that LD50 of AHV-PI was 13.44mg/kg and 95% confidence limit of AHV-PI was 14.16 mg / kg 路3.The body temperature of mice after intraperitoneal injection of AHV-PI decreased by 2 ~ 5 鈩，

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