血压波动性增高对阿司匹林抗血小板作用的影响

发布时间：2018-05-06 15:47

本文选题：血压波动性 + 血小板　；参考：《泰山医学院》2014年硕士论文

【摘要】：目的研究血压波动性(BPV)增高对阿司匹林(Asp)抗血小板作用的影响及机制。方法实验分为在体实验和体外实验两部分。 1.在体实验将Sprague-Dawley(SD)大鼠行去窦弓神经(SAD)或假手术(Sham)，随机分为8组：Sham组、SAD组、Sham+Asp低剂量组(5mg/kg)、SAD+Asp低剂量组(5mg/kg)、Sham+Asp中剂量组(15mg/kg)、SAD+Asp中剂量组(15mg/kg)、Sham+Asp高剂量组(45mg/kg)、SAD+Asp高剂量组(45mg/kg)，n=6。术后四周，在清醒自由活动状态下，检测血压(BP)、BPV和压力感受性反射敏感性(BRS)的变化。Asp每日灌胃给药，Sham组和SAD组大鼠灌胃等体积的溶媒，连续7天后，处死大鼠心脏取血，取肝素抗凝血，利用比浊法测定二磷酸腺苷(ADP)、胶原、凝血酶、花生四烯酸(AA)诱导的血小板聚集率；应用小室灌流技术检测在高切变率(1200s-1)和低切变率(300s-1)下血小板对胶原的黏附率；采用流式细胞技术测定血小板表面P-选择素表达、活性氧(ROS)、一氧化氮(NO)以及钙离子(Ca2+)平均荧光强度；取EDTA-消炎痛抗凝血，放免法检测血浆TXB2含量，AA诱导下测定血小板环氧化酶(COX)活性。 2.体外实验取SD大鼠肝素抗凝血，随机分为8组：非波动组、波动组、非波动+Asp低浓度组(10μg/ml)、波动+Asp低浓度组(10μg/ml)、非波动+Asp中浓度组(30μg/ml)、波动+Asp中浓度组(30μg/ml)、非波动+Asp高浓度组(100μg/ml)、波动+Asp高浓度组(100μg/ml)，n=5。Asp(非波动组、波动组应用等体积溶媒)和血液在37℃孵育15min后，进入体外压力波动模型5min，利用流式细胞技术检测血小板微聚集率和上述相应指标，应用小室灌流技术测定血小板黏附率。结果 1.在体实验结果 1.1SAD大鼠血流动力学和BRS变化与Sham组比较，SAD组大鼠SBP、DBP和MBP无明显差异，但是SBPV、DBPV和MBPV均显著增高，BRS显著降低，表明SAD大鼠呈单纯性BPV增高状态，且动脉压力感受性反射功能明显受损。 1.2血压波动性增高对Asp抗血小板作用的影响①与相应Sham组比较，，SAD(Asp0、5、15mg/kg)组大鼠血小板聚集率、黏附率、血小板表面P-选择素阳性表达率、血浆TXB2含量及COX活性均显著升高；②与Sham+Asp(45mg/kg)组比较，SAD+Asp(45mg/kg)组大鼠血小板聚集率、黏附率、血小板表面P-选择素阳性表达率、COX活性、血浆中TXB2含量均无显著差异；③与SAD+Asp(0mg/kg)组比较，SAD+Asp (45mg/kg)组大鼠上述指标均显著降低。 1.3血小板COX活性及ROS、NO、Ca2+水平变化①与相应Sham组比较，SAD(Asp0、5、15mg/kg)组大鼠血小板COX活性升高，血小板ROS、Ca2+平均荧光强度增高，NO平均荧光强度降低。②与相应Sham+Asp(45mg/kg)组比较，SAD+Asp(45mg/kg)组大鼠血小板COX活性以及ROS、Ca2+、NO平均荧光强度无显著差异；③与SAD+Asp(0mg/kg)组比较，SAD+Asp(45mg/kg)组大鼠血小板COX活性以及ROS、Ca2+平均荧光强度降低，NO平均荧光强度升高。 2.体外实验结果 2.1压力和压力波动性变化与非波动组比较，波动组的SBP、DBP和MBP无明显差异，但SBPV、DBPV和MBPV均显著增高。表明模拟SAD大鼠BPV增高的体外模型是成功的。 2.2压力波动性增高对阿司匹林抗血小板作用的影响①与相应非波动组比较，波动组(Asp0、10、30μg/ml)血小板表面P-选择素阳性表达率、微聚集率和黏附率均显著升高；②与非波动+Asp(100μg/ml)组比较，波动+Asp(100μg/ml)组血小板表面P-选择素阳性表达率、微聚集率和黏附率均无显著差异；③与波动+Asp(0μg/ml)组比较，波动+Asp(100μg/ml)组血小板表面P-选择素阳性表达率、微聚集率和黏附率均显著降低。 2.3血小板ROS、NO、Ca2+探针荧光强度的变化①与相应非波动组比较，波动组(Asp0、10、30μg/ml)血小板ROS、Ca2+平均荧光强度增高，NO的平均荧光强度降低；②与非波动+Asp(100μg/ml)组比较，波动+Asp(100μg/ml)组血小板ROS、Ca2+、NO平均荧光强度无显著差异；③与波动+Asp(0μg/ml)组比较，波动+Asp(100μg/ml)组血小板ROS、Ca2+平均荧光强度显著降低，NO平均荧光强度明显升高。结论血压波动性增高能够降低阿司匹林对血小板活化的抑制作用，机制与血小板COX活性增高、氧化应激和NO、Ca2+信号变化有关。增加阿司匹林的剂量可以对抗血压波动性增高的这种作用。
[Abstract]:objective
Objective to study the effect and mechanism of increased blood pressure (BPV) on antiplatelet action of aspirin (Asp).
Method
The experiment is divided into two parts: in vivo experiment and in vitro experiment.
1. Sprague-Dawley (SD) rats were randomly divided into 8 groups: Sham group, SAD group, SAD group, Sham+Asp low dose group (5mg/kg), SAD+Asp low dose group (5mg/kg), Sham+Asp medium dose group (15mg/kg), high dose group, high dose group. In the latter four weeks, the changes of blood pressure (BP), BPV, and pressure receptive reflex sensitivity (BRS) were measured in the wake of conscious and free activity..Asp was administered daily to the gastric administration, the Sham group and the SAD group were given the volume of the solvent. After 7 days, the rat heart was killed and the heparin anticoagulant blood was taken, and the ADP, collagen, thrombin and flower were measured by turbidimetry. The platelet aggregation rate induced by four enoic acid (AA); the adhesion rate of platelets to collagen at high shear rate (1200s-1) and low shear rate (300s-1) was detected by small ventricular perfusion technique. Flow cytometry was used to determine the expression of P- selectin on the surface of platelets, reactive oxygen species (ROS), nitric oxide (NO) and calcium ion (Ca2+) average fluorescence intensity, and E DTA- indomethacin anticoagulant, radioimmunoassay was used to detect plasma TXB2 content, and AA was used to determine platelet activity of COX.
2. the heparin anticoagulant of SD rats was randomly divided into 8 groups: non wave group, wave group, non wave +Asp low concentration group (10 u g/ml), fluctuating +Asp low concentration group (10 mu g/ml), non fluctuating +Asp concentration group (30 mu g/ml), undulating +Asp concentration group (30 u g/ml), non wave +Asp high concentration group (100 mu g/ml), undulant +Asp high concentration group (100 mu), 100 (non wave group, wave group and other volume solvent) and blood were incubated at 37 centigrade for 15min to enter the pressure wave model 5min in vitro. The platelet aggregation rate and the corresponding index were detected by flow cytometry, and the platelet adhesion rate was measured by small chamber perfusion technique.
Result
1. in vivo experiment results
The changes of hemodynamics and BRS in 1.1SAD rats were compared with that of group Sham. There was no significant difference in SBP, DBP and MBP in group SAD, but SBPV, DBPV and MBPV were all significantly increased, BRS significantly decreased, indicating that SAD rats showed a state of pure increase, and the arterial pressure receptive reflex was significantly impaired.
1.2 the effect of high fluctuation of blood pressure on the antiplatelet effect of Asp (1) compared with the corresponding Sham group, the platelet aggregation rate, adhesion rate, the positive expression of P- selectin on the platelet surface, the plasma TXB2 content and the COX activity were significantly increased in the SAD (Asp0,5,15mg/kg) group, and the platelet aggregation in the SAD+Asp (45mg/kg) group was compared with the Sham+Asp (45mg/kg) group. There was no significant difference in the rate of collection, adhesion, the positive expression of P- selectin on the platelet surface, the activity of COX, and the content of TXB2 in the plasma. (3) compared with the SAD+Asp (0mg/kg) group, the above indexes in the SAD+Asp (45mg/kg) group were significantly reduced.
1.3 the activity of platelet COX and the changes of ROS, NO, Ca2+ level 1. Compared with the corresponding Sham group, the activity of platelet COX in the SAD (Asp0,5,15mg/kg) group increased, the platelet ROS, the average fluorescence intensity of Ca2+ increased, and the NO average fluorescence intensity decreased. There was no significant difference in the average fluorescence intensity. Compared with the SAD+Asp (0mg/kg) group, the COX activity of platelets in the rats of SAD+Asp (45mg/kg) group and the average fluorescence intensity of Ca2+ decreased and the average fluorescence intensity of NO increased.
2. in vitro experimental results
2.1 there was no significant difference in SBP, DBP and MBP in the fluctuation group, but the SBPV, DBPV and MBPV were all significantly higher than those in the non wave group, indicating that the model in vitro of the increased BPV in the simulated SAD rats was successful.
2.2 the effect of increased pressure fluctuation on the antiplatelet action of aspirin (1) compared with the non wave group, the positive expression rate of P- selectin on the platelet surface, the rate of micro aggregation and adhesion of the platelet surface in the wave group (Asp0,10,30 mu g/ml) were all significantly increased. 2. Compared with the non wave +Asp (100 mu g/ml) group, the platelet surface P- selectin in the group of +Asp (100 mu g/ml) group was fluctuated. There was no significant difference in positive expression rate, micro aggregation rate and adhesion rate. Compared with the wave +Asp (0 mu g/ml) group, the positive expression rate of P- selectin on the platelets on the fluctuating +Asp (100 g/ml) group decreased significantly.
2.3 the changes in the fluorescence intensity of the platelet ROS, NO, and Ca2+ probe (1) compared with the non wave group, the platelet ROS in the wave group (Asp0,10,30 mu g/ml), the average fluorescence intensity of Ca2+ increased and the average fluorescence intensity of NO decreased, and there was no significant difference in the average fluorescence intensity of the fluctuation +Asp (100 micron g/ml) group compared with the non undulation +Asp (100 micron g/ml) group. (3) compared with the fluctuating +Asp (0 g/ml) group, the mean fluorescence intensity of platelet ROS and Ca2+ decreased significantly in the fluctuating +Asp (100 g/ml) group, and the average fluorescence intensity of NO increased significantly.
conclusion
High blood pressure fluctuation can reduce the inhibitory effect of aspirin on platelet activation. The mechanism is associated with increased platelet COX activity, oxidative stress and changes in NO and Ca2+ signals. The increase of aspirin dose can antagonism the effect of high blood pressure fluctuation.

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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