蛇毒血小板抑制因子對(duì)MIRI大鼠血流變的影響及機(jī)制研究
發(fā)布時(shí)間：2018-10-23 12:29
【摘要】：目的:探討皖南蝮蛇毒血小板抑制因子(Agkistrodon halys venom platelet inhibitor,AHV-PI)對(duì)心肌缺血再灌注損傷(myocardial ischemia reperfusion injury,MIRI)模型大鼠血液流變學(xué)變化的影響并分析其可能的作用機(jī)制。方法:1.實(shí)驗(yàn)動(dòng)物分組:SD雄性大鼠30只,隨機(jī)分為假手術(shù)組、心肌缺血再灌注損傷模型組和AHV-PI實(shí)驗(yàn)組,AHV-PI實(shí)驗(yàn)組根據(jù)所給AHV-PI的劑量(0.05,0.1,0.2mg/kg)再分三組,每組6只。2.模型制備:首先通過(guò)結(jié)扎大鼠左冠狀動(dòng)脈前降支方法制備大鼠心肌缺血再灌注損傷(MIRI)模型同時(shí)假手術(shù)組僅開胸不復(fù)制該模型,AHV-PI各實(shí)驗(yàn)組在造模前經(jīng)舌下靜脈注射相應(yīng)劑量AHV-PI預(yù)處理。RM6240生物信號(hào)采集處理系統(tǒng)監(jiān)測(cè)所有大鼠心電變化,實(shí)驗(yàn)完成后立即處死大鼠,取其心臟組織,利用HE染色法觀察大鼠心臟形態(tài)學(xué)變化。3.指標(biāo)檢測(cè):各實(shí)驗(yàn)組大鼠頸總動(dòng)脈取血8 m L,分別利用錐板式血液流變學(xué)分析儀測(cè)定其全血高切、中切和低切粘度及血漿粘度;采用血栓彈力圖儀(TEG)分析其凝血時(shí)間(R)、血凝塊形成時(shí)間(K)、Alpha角度(A)和凝血最大幅度(MA),采用比濁法檢測(cè)血小板一分鐘聚集率(A1)、三分鐘聚集率(A3)、五分鐘聚集率(A5)、最大聚集時(shí)間(Tmax)和聚集幅度。取血漿以酶聯(lián)免疫吸附法(ELISA)檢測(cè)血漿v WF、P-選擇素的濃度變化。蛋白質(zhì)免疫印跡法(Western bloting)觀測(cè)AHV-PI干預(yù)下,大鼠血小板膜糖蛋白GPVI的表達(dá)水平變化。結(jié)果:1.假手術(shù)組心率增快;MIRI模型組心電顯示心臟缺血后心電圖顯示心率減慢,S-T段向上抬高,T波亦有所提高。各實(shí)驗(yàn)組大鼠心肌組織形態(tài)改變:病理切片顯示AHV-PI中劑量實(shí)驗(yàn)組(0.1 mg/kg)、AHV-PI高劑量實(shí)驗(yàn)組(0.2 mg/kg)和假手術(shù)組心肌無(wú)腫脹,心肌細(xì)胞排列較整齊,無(wú)局部變性。MIRI模型組和AHV-PI低劑量實(shí)驗(yàn)組(0.05mg/kg)大鼠心肌腫脹,核固縮、破碎、溶解。2.AHV-PI對(duì)MIRI大鼠血液流變學(xué)變化的影響:與MIRI模型組比較,AHV-PI中劑量實(shí)驗(yàn)組和高劑量實(shí)驗(yàn)組全血高切、中切和低切粘度及血漿粘度明顯降低(P0.05),R值和K值明顯延長(zhǎng)(P0.05),A和MA減小(P0.05),血小板一分鐘聚集率(A1)、三分鐘聚集率(A3)、五分鐘聚集率(A5)、最大聚集時(shí)間(Tmax)和聚集幅度均明顯降低(P0.05);而和假手術(shù)組相比較并無(wú)顯著差異。3.AHV-PI對(duì)MIRI大鼠血小板活性的影響:與MIRI模型組比較,AHV-PI中劑量實(shí)驗(yàn)組和高劑量實(shí)驗(yàn)組血漿v WF、P-選擇素濃度、GPVI的表達(dá)水平亦明顯降低(P0.05),而AHV-PI中劑量實(shí)驗(yàn)組和高劑量實(shí)驗(yàn)組較假手術(shù)組上述各項(xiàng)指標(biāo)均無(wú)明顯改變(P0.05)。其中MIRI模型組與AHV-PI低劑量組之間、AHV-PI中劑量組與AHV-PI高劑量組之間上述指標(biāo)亦無(wú)明顯差異(P0.05)。結(jié)論:1.AHV-PI中劑量實(shí)驗(yàn)組(0.1 mg/kg)和AHV-PI高劑量實(shí)驗(yàn)組(0.2 mg/kg)明顯減輕大鼠心肌缺血再灌注損傷;2.AHV-PI可以顯著改善心肌缺血再灌注損傷引起的血液流變學(xué)變化;3.AHV-PI減輕心肌缺血再灌注損傷后血液高凝狀態(tài)的機(jī)制可能是通過(guò)降低v WF、P-選擇素釋放和抑制GPVI的表達(dá)。
[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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R54
本文編號(hào)：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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R54
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