【作者】 余瑞铭；

【导师】 许云禄；

【作者基本信息】 福建医科大学 ， 药理学， 2008， 硕士

【摘要】 蛇毒是由多种生物活性多肽和蛋白质组成的混合物,由于蛇毒蛋白酶可从凝血、纤溶及血小板聚集等多环节影响血液系统功能[1],因此从蛇毒中研发治疗血栓性疾病的药物,成为国内外研究的热点之一。1993年,张云首次从竹叶青蛇毒中分离纯化出蛇毒纤溶酶原激活剂(TSV-PA),为从蛇毒中开发新型抗栓药物提供新的课题,本课题旨在从江浙产短尾蝮( Gloydius brevicaudus )蛇毒中分离纯化出纤溶酶原激活剂(plasminogen activator from Gloydius brevicaudus venom, GBV-PA),并观察其对动物实验性血栓预防和治疗作用。1.GBV-PA的分离纯化应用苯甲脒琼脂糖凝胶6B (Benzamidine Sepharose 6B)亲和层析去除短尾蝮蛇毒中大部分的非目的蛋白,目的组分再经Lichrospher C18 4.6/250反相层析柱,在Waters 600E高效液相色谱仪上进行纯化,获得具有间接溶解纤维蛋白作用的第Ⅱ峰,即GBV-PA。GBV-PA经SDS-PAGE鉴定,在β-巯基乙醇还原和非还原条件下,均显示单一条带,提示其为单一肽链的蛋白质,分子量约为34kDa。2.GBV-PA对FeC13诱导的大鼠颈总动脉血栓的溶栓作用以FeC13诱导大鼠颈总动脉血栓形成,以大鼠舌根温度的变化判定血栓的形成和溶解情况。结果大鼠静脉注射GBV-PA150和300μg·kg-1,可以明显提高为FeC13所降低的大鼠舌根温度,该作用持续420min以上;UK2000IU·kg-1静注也能明显提高大鼠舌根温度,但180min后作用开始减弱。比较各组大鼠颈总动脉血栓重量,GBV-PA150和300μg·kg-1组血栓重量分别为25.3±0.06mg和17.6±0.73mg,尿激酶组为22.9±0.73mg,与生理盐水对照组(30.8±2.62mg)相比均有显著性差异(p<0.01),说明GBV-PA对大鼠颈总动脉血栓具有治疗作用,且作用持续时间比尿激酶更长。3.GBV-PA对FeC13诱导的大鼠颈总动脉混合性血栓的抑制作用大鼠颈总动脉血栓模型的制备方法同前,预先静脉注射GBV-PA150和300μg·kg-1,大鼠舌根温度下降值明显小于阴性对照组(p<0.05)。GBV-PA还能明显减轻血栓重量,GBV-PA150和300μg·kg-1组血栓重量分别为21.5±0.13mg和15.2±0.69mg,尿激酶组为20.6±0.33mg,与生理盐水对照组(31.6±0.67mg)比较均有显著差别(p<0.05)。说明GBV-PA可抑制FeC13诱导的大鼠颈总动脉血栓的形成。4.GBV-PA对大鼠颈动静脉(A—V)回路形成的血小板性血栓的抑制作用大鼠颈动静脉(A—V)回路形成后,血流中的血小板接触旁路循环中的丝线粗糙面时,发生黏附、聚集,血小板聚集物环绕丝线表面形成血栓。大鼠静脉注射GBV-PA150和300μg·kg-1能明显减轻血栓重量,GBV-PA150和300μg·kg-1组血栓重量分别为23.3±1.57mg和14.7±0.66mg,阿司匹林组为14.9±0.90mg,与生理盐水对照组(47.1±1.27mg)比较均有显著差别(p<0.01)。说明GBV-PA对血小板性动脉血栓的形成具有明显抑制效应。5.GBV-PA对兔脑粉浸液诱发的大鼠下腔静脉血栓形成的影响兔脑粉悬液富含组织因子,注入下腔静脉后启动外源性凝血途径,诱导血栓形成。大鼠静脉注射GBV-PA150和300μg·kg-1能明显减轻血栓重量,GBV-PA150和300μg·kg-1组血栓重量分别为20.3±2.15mg和12.5±1.44mg,阿司匹林组为16.2±3.15mg,与生理盐水对照组(52.3±12.3mg)比较均有显著差别(p<0.01)。说明GBV-PA可预防大鼠下腔静脉血栓的形成。6.GBV-PA对ADP-Na2诱导的大鼠急性肺栓塞的影响由ADP诱导血小板聚集形成的微小血栓,随同血液流经肺动脉时,造成肺动脉的微栓塞,出现呼吸窘迫征,大鼠出现呼吸喘促等症状。记录大鼠呼吸喘促的持续时间,GBV-PA150和300μg·kg-1组分别为189.3±12.4s和131.2±12.0s,阿司匹林组为159.9±14.2s,与生理盐水对照组(918.2±82.4s)比较均有显著差别(p<0.01)。说明GBV-PA可明显缩短ADP诱导的血小板聚集性肺栓塞引起呼吸喘促的时间。7.GBV-PA对实验性犬急性脑梗塞的治疗作用采用介入技术建立比格犬急性大脑中动脉栓塞模型,栓塞后行数字减影脑血管造影观察所栓塞血管的再通情况。19只比格犬随机分为GBV-PA 18.75、37.5和75μg·kg-1低、中和高剂量组,阳性对照组为104IU·kg-1剂量的rt-PA,阴性对照组为生理盐水。1小时rt-PA组、GBV-PA低剂量、中剂量和高剂量组的血管再通率分别为100%(2/2)、0%(0/4)、25%(1/4)和50%(2/4), 2小时各组的再通率分别为100%、0%、50%和75%,3小时各组的再通率分别为100%、50%、75%和100%,生理盐水组3h内均未发现血管再通。说明GBV-PA各剂量组的血管再通率存在明显的量效和时效关系,随着用药剂量的增大和药物作用时间的延长,血管再通率上升。结论:大鼠iv GBV-PA150和300μg·kg-1均可显著促进为FeC13诱导的颈总动脉血栓的消退,且作用持续时间比尿激酶更长;大鼠iv GBV-PA150和300μg·kg-1均可抑制FeC13诱导的大鼠颈总动脉血栓的形成;大鼠iv GBV-PA150、300μg·kg-1均可显著抑制大鼠颈动静脉回路产生的血小板性血栓的形成;大鼠iv GBV-PA150、300μg·kg-1均可显著抑制兔脑粉诱发的大鼠下腔静脉血栓的形成;GBV-PA在体内能明显对抗ADP诱导的大鼠体内血小板聚集;犬动脉内输注GBV-PA18.75、37.5和75μg·kg-1对实验性犬急性脑梗塞具有治疗作用,血管再通率存在明显的量效和时效关系。更多还原

【Abstract】 Snake venoms are complex mixtures containing many different biologically active proteins and peptides. A number of these proteins interact with components of the human hemostatic system.In 1993,A novel plasminogen activator form Trimeresurus stejnegeri venom(TSV—PA) has been identified and purified by Yun Zhang for the first time.It offers the new subject for developing new medicine from venom.This study was designed to isolate and purify novel plasminogen activator from Gloydius brevicaudus venom(GBV)and analyse its characterization and biological activities.1. Purification of the plasminogen activator from Gloydius brevicaudus Venom. Affinity chromatography of Gloydius brevicaudus Venom in Benzamidine Sepharose 6B resulted in the separation of two protein peaks. GBV-PA was purified by Lichrospher C18 4.6/250 reverse chromatography. The fractionⅡcan specifically activate plasminogen through an enzymatic reaction estimated by the fibrin plate method. It was homogeneous as a single band showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE,Tris system).2. Thrombolytic effect of GBV-PA in rats common carotid artery of thrombosis. Thrombogenesis was induced by FeC13, thrombogenesis and thrombolysis were judged by temperature of lingual root of rats. GBV-PA 150、300μg·kg-1 iv could upgrade the temperature of lingual root of rats, the effect continued more than 420 minutes; UK 2000IU·kg-1 iv also upgraded the temperature of lingual root of rats , but the effect attenuated after 180 minutes.Comparing with the thrombus weight, GBV-PA 150、300μg·kg-1 were 25.3±0.06mg and 17.6±0.73mg respectively,UK was 22.9±0.73mg,with obvious difference compared with NS (p<0.01). GBV-PA has the thrombolytic effect to thrombus of rats common carotid artery, and persists longer time compared with UK.3. Protective effect of GBV-PA in rats common carotid artery of thrombosis . Thrombogenesis was induced by FeC13 .The drug was given 60 minutes before spreading filter paper of FeC13 . GBV-PA 150、300μg·kg-1 iv could inhibit the degrad of temperature of rats lingual root. Comparing with the thrombus weight, GBV-PA 150、300μg·kg-1 were 21.5±0.13mg and 15.2±0.69mg respectively,UK was 20.6±0.33mg,with obvious difference compared with NS (p<0.05). GBV-PA has the inhibition to thrombosis of rats common carotid artery.4. Effect of GBV-PA on thrombolysis of rats in man-made artery-vein circulation.The effect of GBV-PA was estimated by weight of thrombus.GBV-PA 150、300μg·kg-1 iv could significantly reduce the weight of thrombus.Comparing with the thrombus weight, GBV-PA 150、300μg·kg-1 were 23.3±1.57mg and 14.7±0.66mg respectively,asprin was 14.9±0.90mg,with obvious difference compared with NS (p<0.01). GBV-PA has the inhibition to thrombosis of rats common carotid artery in man-made artery-vein circulation.5. Thrombolytic effect of GBV-PA on thrombus of inferior caval vein induced by power of rabbit brain.The thrombus of inferior caval vein was induced by power of rabbit brain. GBV-PA 150、300μg·kg-1 iv could significantly reduce the weight of thrombus.Comparing with the thrombus weight, GBV-PA 150、300μg·kg-1 were 20.3±2.15mg and 12.5±1.44mg respectively,asprin was 16.2±3.15mg,with obvious difference compared with NS (p<0.01). GBV-PA has the inhibition to thrombosis of rats inferior caval vein induced by power of rabbit brain.6. Protective effect of GBV-PA on rats with pulmonary thrombus.We observed the effect on the respiratory distress due to pulmonary thrombosis induced by ADP-Na2 after GBV-PA 150 and 300μg·kg-1 iv in rats.Comparing with time of respiratory distress, GBV-PA 150、300μg·kg-1 were 189.3±12.4s and 131.2±12.0s respectively,asprin was 159.9±14.2s,with obvious difference compared with NS (p<0.01). GBV-PA markedly relieved the respiratory distress.7. Thrombolytic effect of GBV-PA on dogs with cerebral thrombus.The model of internal cerebral embolism was established with interventional technique in 19 beagle adult dogs,which were randomly divided into 5 groups including control group, group of 104IU·kg-1 dose of rt-PA, group of 18.75μg·kg-1 dose,37.5μg·kg-1 dose,75μg·kg-1 dose of GBV-PA. Angiography was performed half,1,2 and 3 hours after thrombolysis to observe recanalization. The rate of recanalization 1 hour after administration of rt-PA,low dose,middle dose,high dose of GBV-PA were 100%,0%,25%and 50%;2 hours were 100%,0%,50% and 75%;3 hours were 100%,50%,75% and 100%,showed significanlly statistic difference compared with control group(p<0.05). There were time-response and dose-response relationships when the cerebral infarction beagle dogs were treated with GBV-PA. ConclusionsGBV-PA 150、300μg·kg-1 iv have the thrombolytic effect to thrombus of rats common carotid artery induced by FeC13, and persists longer time compared with UK.;GBV-PA 150、300μg·kg-1 iv have the inhibition to thrombosis of rats common carotid artery induced by FeC13; GBV-PA 150、300μg·kg-1 iv have the inhibition to thrombosis of rats common carotid artery in man-made artery-vein circulation; GBV-PA 150、300μg·kg-1 iv have the inhibition to thrombosis of rats inferior caval vein induced by power of rabbit brain; GBV-PA 150、300μg·kg-1 iv relieve the respiratory distress; GBV-PA 150、300μg·kg-1 iv have thrombolytic effect to beagle dogs with cerebral thrombus, there are time-response and dose-response relationships when the cerebral infarction beagle dogs are treated with GBV-PA.更多还原