蛇毒血小板抑制因子对MIRI大鼠血流变的影响及机制研究
发布时间：2018-10-23 12:29
【摘要】：目的:探讨皖南蝮蛇毒血小板抑制因子(Agkistrodon halys venom platelet inhibitor,AHV-PI)对心肌缺血再灌注损伤(myocardial ischemia reperfusion injury,MIRI)模型大鼠血液流变学变化的影响并分析其可能的作用机制。方法:1.实验动物分组:SD雄性大鼠30只,随机分为假手术组、心肌缺血再灌注损伤模型组和AHV-PI实验组,AHV-PI实验组根据所给AHV-PI的剂量(0.05,0.1,0.2mg/kg)再分三组,每组6只。2.模型制备:首先通过结扎大鼠左冠状动脉前降支方法制备大鼠心肌缺血再灌注损伤(MIRI)模型同时假手术组仅开胸不复制该模型,AHV-PI各实验组在造模前经舌下静脉注射相应剂量AHV-PI预处理。RM6240生物信号采集处理系统监测所有大鼠心电变化,实验完成后立即处死大鼠,取其心脏组织,利用HE染色法观察大鼠心脏形态学变化。3.指标检测:各实验组大鼠颈总动脉取血8 m L,分别利用锥板式血液流变学分析仪测定其全血高切、中切和低切粘度及血浆粘度;采用血栓弹力图仪(TEG)分析其凝血时间(R)、血凝块形成时间(K)、Alpha角度(A)和凝血最大幅度(MA),采用比浊法检测血小板一分钟聚集率(A1)、三分钟聚集率(A3)、五分钟聚集率(A5)、最大聚集时间(Tmax)和聚集幅度。取血浆以酶联免疫吸附法(ELISA)检测血浆v WF、P-选择素的浓度变化。蛋白质免疫印迹法(Western bloting)观测AHV-PI干预下,大鼠血小板膜糖蛋白GPVI的表达水平变化。结果:1.假手术组心率增快;MIRI模型组心电显示心脏缺血后心电图显示心率减慢,S-T段向上抬高,T波亦有所提高。各实验组大鼠心肌组织形态改变:病理切片显示AHV-PI中剂量实验组(0.1 mg/kg)、AHV-PI高剂量实验组(0.2 mg/kg)和假手术组心肌无肿胀,心肌细胞排列较整齐,无局部变性。MIRI模型组和AHV-PI低剂量实验组(0.05mg/kg)大鼠心肌肿胀,核固缩、破碎、溶解。2.AHV-PI对MIRI大鼠血液流变学变化的影响:与MIRI模型组比较,AHV-PI中剂量实验组和高剂量实验组全血高切、中切和低切粘度及血浆粘度明显降低(P0.05),R值和K值明显延长(P0.05),A和MA减小(P0.05),血小板一分钟聚集率(A1)、三分钟聚集率(A3)、五分钟聚集率(A5)、最大聚集时间(Tmax)和聚集幅度均明显降低(P0.05);而和假手术组相比较并无显著差异。3.AHV-PI对MIRI大鼠血小板活性的影响:与MIRI模型组比较,AHV-PI中剂量实验组和高剂量实验组血浆v WF、P-选择素浓度、GPVI的表达水平亦明显降低(P0.05),而AHV-PI中剂量实验组和高剂量实验组较假手术组上述各项指标均无明显改变(P0.05)。其中MIRI模型组与AHV-PI低剂量组之间、AHV-PI中剂量组与AHV-PI高剂量组之间上述指标亦无明显差异(P0.05)。结论:1.AHV-PI中剂量实验组(0.1 mg/kg)和AHV-PI高剂量实验组(0.2 mg/kg)明显减轻大鼠心肌缺血再灌注损伤;2.AHV-PI可以显著改善心肌缺血再灌注损伤引起的血液流变学变化;3.AHV-PI减轻心肌缺血再灌注损伤后血液高凝状态的机制可能是通过降低v WF、P-选择素释放和抑制GPVI的表达。
[Abstract]:Aim: to investigate the effect of platelet inhibitor (Agkistrodon halys venom platelet inhibitor,AHV-PI (Agkistrodon halys venom platelet inhibitor,AHV-PI) from Agkistrodon halys venom on hemorheology in rats with myocardial ischemia-reperfusion injury (myocardial ischemia reperfusion injury,MIRI) and to analyze its possible mechanism. Methods: 1. The experimental animals were divided into three groups: sham operation group, myocardial ischemia-reperfusion injury model group and AHV-PI experimental group. The experimental group of AHV-PI was divided into three groups according to the dosage of AHV-PI (0.05g / kg, 0.1g / kg), 6 rats in each group. Model preparation: the (MIRI) model of myocardial ischemia-reperfusion injury was established by ligating the anterior descending branch of left coronary artery in rats. The model was not reproduced in the sham operation group. The experimental groups of AHV-PI were injected through sublingual vein before modeling. The corresponding dose of AHV-PI pretreatment. The RM6240 biological signal acquisition and processing system monitored the changes of ECG in all rats. The rats were killed immediately after the experiment and their heart tissues were taken. The morphologic changes of the heart were observed by HE staining. The blood samples were collected from the common carotid artery of rats in each experimental group for 8 mL. The whole blood high shear viscosity, middle shear viscosity and low shear viscosity and plasma viscosity were measured by cone-plate hemorheology analyzer. Thromboelastography (TEG) was used to analyze the clotting time, (R), clot formation time, (K), Alpha angle and maximum (MA),. The platelet aggregation rate (A1), three-minute aggregation rate (A3), five-minute aggregation rate (A5) and maximum aggregation rate were detected by turbidimetric method in one minute (A1), three minutes (A3), five minutes (A5), and the maximum aggregation rate (A5). Set time (Tmax) and aggregation amplitude. The concentration of v WF,P- selectin in plasma was detected by enzyme linked immunosorbent assay (ELISA). Western blot (Western bloting) was used to detect the expression of glycoprotein GPVI in rat platelets after AHV-PI intervention. The result is 1: 1. The ECG of MIRI model group showed that the heart rate slowed down, the S-T segment raised upward and the T wave increased. Morphologic changes of myocardium in each experimental group: pathological sections showed that there was no swelling of myocardium in the middle dose group of AHV-PI (0. 1 mg/kg), the high dose group of AHV-PI (0. 2 mg/kg) and the group of sham operation, and the myocardial cells were arranged neatly. No local denaturation. Myocardial swelling, nuclear contraction, fragmentation and dissolution in MIRI model group and AHV-PI low dose experimental group (0.05mg/kg). Effects of 2.AHV-PI on hemorheology in MIRI rats: compared with MIRI model group, AHV-PI medium dose experimental group and high dose experimental group had high whole blood shear. Middle and low shear viscosity and plasma viscosity were significantly decreased (P0.05), R and K values were significantly prolonged (P0.05), A and MA decreased P0.05), platelet aggregation rate in one minute (A1), three-minute aggregation rate (A3), five-minute aggregation rate (A5), maximum aggregation time (Tmax) and aggregation range were significant. The effect of 3.AHV-PI on platelet activity of MIRI rats: compared with MIRI model group, the plasma v WF,P- selectin concentration and GPVI expression level of AHV-PI medium dose group and high dose experimental group were also clear. Significantly decreased (P0.05), while AHV-PI in the middle dose experimental group and high dose experimental group compared with the sham-operation group, the above indexes were not significantly changed (P0.05). There was no significant difference between the MIRI model group and the AHV-PI low dose group, the AHV-PI medium dose group and the AHV-PI high dose group (P0.05). Conclusion: 1.AHV-PI medium dose group (0. 1 mg/kg) and AHV-PI high dose group (0. 2 mg/kg) can significantly reduce myocardial ischemia-reperfusion injury in rats, 2.AHV-PI can significantly improve the hemorheological changes induced by myocardial ischemia reperfusion injury, and 3.AHV-PI can alleviate myocardial ischemia reperfusion injury. The mechanism of hypercoagulability after perfusion injury may be by reducing the release of v WF,P- selectin and inhibiting the expression of GPVI.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
本文编号：2289241
[Abstract]:Aim: to investigate the effect of platelet inhibitor (Agkistrodon halys venom platelet inhibitor,AHV-PI (Agkistrodon halys venom platelet inhibitor,AHV-PI) from Agkistrodon halys venom on hemorheology in rats with myocardial ischemia-reperfusion injury (myocardial ischemia reperfusion injury,MIRI) and to analyze its possible mechanism. Methods: 1. The experimental animals were divided into three groups: sham operation group, myocardial ischemia-reperfusion injury model group and AHV-PI experimental group. The experimental group of AHV-PI was divided into three groups according to the dosage of AHV-PI (0.05g / kg, 0.1g / kg), 6 rats in each group. Model preparation: the (MIRI) model of myocardial ischemia-reperfusion injury was established by ligating the anterior descending branch of left coronary artery in rats. The model was not reproduced in the sham operation group. The experimental groups of AHV-PI were injected through sublingual vein before modeling. The corresponding dose of AHV-PI pretreatment. The RM6240 biological signal acquisition and processing system monitored the changes of ECG in all rats. The rats were killed immediately after the experiment and their heart tissues were taken. The morphologic changes of the heart were observed by HE staining. The blood samples were collected from the common carotid artery of rats in each experimental group for 8 mL. The whole blood high shear viscosity, middle shear viscosity and low shear viscosity and plasma viscosity were measured by cone-plate hemorheology analyzer. Thromboelastography (TEG) was used to analyze the clotting time, (R), clot formation time, (K), Alpha angle and maximum (MA),. The platelet aggregation rate (A1), three-minute aggregation rate (A3), five-minute aggregation rate (A5) and maximum aggregation rate were detected by turbidimetric method in one minute (A1), three minutes (A3), five minutes (A5), and the maximum aggregation rate (A5). Set time (Tmax) and aggregation amplitude. The concentration of v WF,P- selectin in plasma was detected by enzyme linked immunosorbent assay (ELISA). Western blot (Western bloting) was used to detect the expression of glycoprotein GPVI in rat platelets after AHV-PI intervention. The result is 1: 1. The ECG of MIRI model group showed that the heart rate slowed down, the S-T segment raised upward and the T wave increased. Morphologic changes of myocardium in each experimental group: pathological sections showed that there was no swelling of myocardium in the middle dose group of AHV-PI (0. 1 mg/kg), the high dose group of AHV-PI (0. 2 mg/kg) and the group of sham operation, and the myocardial cells were arranged neatly. No local denaturation. Myocardial swelling, nuclear contraction, fragmentation and dissolution in MIRI model group and AHV-PI low dose experimental group (0.05mg/kg). Effects of 2.AHV-PI on hemorheology in MIRI rats: compared with MIRI model group, AHV-PI medium dose experimental group and high dose experimental group had high whole blood shear. Middle and low shear viscosity and plasma viscosity were significantly decreased (P0.05), R and K values were significantly prolonged (P0.05), A and MA decreased P0.05), platelet aggregation rate in one minute (A1), three-minute aggregation rate (A3), five-minute aggregation rate (A5), maximum aggregation time (Tmax) and aggregation range were significant. The effect of 3.AHV-PI on platelet activity of MIRI rats: compared with MIRI model group, the plasma v WF,P- selectin concentration and GPVI expression level of AHV-PI medium dose group and high dose experimental group were also clear. Significantly decreased (P0.05), while AHV-PI in the middle dose experimental group and high dose experimental group compared with the sham-operation group, the above indexes were not significantly changed (P0.05). There was no significant difference between the MIRI model group and the AHV-PI low dose group, the AHV-PI medium dose group and the AHV-PI high dose group (P0.05). Conclusion: 1.AHV-PI medium dose group (0. 1 mg/kg) and AHV-PI high dose group (0. 2 mg/kg) can significantly reduce myocardial ischemia-reperfusion injury in rats, 2.AHV-PI can significantly improve the hemorheological changes induced by myocardial ischemia reperfusion injury, and 3.AHV-PI can alleviate myocardial ischemia reperfusion injury. The mechanism of hypercoagulability after perfusion injury may be by reducing the release of v WF,P- selectin and inhibiting the expression of GPVI.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
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