【作者】 杜文涛；

【导师】 夏岳；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:目前,冠心病尤其是急性心肌梗死(AMI),已成为危害人类健康的主要疾病之一。急性心肌梗死发生后,心脏的收缩和舒张功能受损,进而出现心力衰竭的症状和体征,发展为心力衰竭。近年来研究发现心肌梗死后神经内分泌系统的激活,在其向心衰发展过程中起到了决定性的作用:一方面该系统的适度激活可以代偿因梗死区局部心肌坏死、舒缩功能丧失而出现的不利的血流动力学变化,可使血压稳定,重要生命器官得到足够的血液供应;同时过度的激活,则可启动心肌细胞凋亡和心室重塑的过程,并可与后二者形成一个恶性循环,逐渐使心肌梗死后的心脏发展为心力衰竭。所以,探讨急性心肌梗死后神经内分泌系统激活和演变规律,对于心肌梗死的积极诊治具有重要的意义。作为神经内分泌系统中重要的成员,脑钠肽和内皮功能在心血管系统中具有重要而又独特的作用。本研究旨在通过动物实验发现急性心肌梗死后大鼠脑钠肽(BNP)、内皮素-1(ET-1)和一氧化氮(NO)的变化规律,以及血管紧张素转换酶抑制剂(卡托普利)在干预这些体液因子方面的作用,为心肌梗死的诊治提供新的理论依据。方法: Wistar大鼠30只,13-16周龄,体重250克左右,均为雄性。术前大鼠被随机分为①对照组(sham group,n=10);②单纯心肌梗死组(AMI group,n=10)③心肌梗死+卡托普利组(AMI+capt group,n=10)。手术过程:大鼠经10%水合氯醛麻醉(35mg/kg腹腔内注射)后,固定于手术台上。行气管插管并连接呼吸机,见大鼠胸廓随呼吸机频率起伏后证明插管成功。设定呼吸参数。75%酒精消毒切口部分,于胸骨左侧做与胸骨平行的纵形切口,约1~1.5cm。分离打开各层肌肉,暴露心脏,挤破心包,迅速弹出心脏,暴露心脏的左室游离壁。在左心耳与肺动脉圆椎之间找到左冠状动脉,在动脉起始点下方2~3mm处,用6-0号丝线结扎左冠状动脉前降支,结扎深度0.3~0.5mm。将心脏送回胸腔,用注射器抽出胸腔内气体,以恢复负压,关闭胸腔,此步骤30s内完成。术中经肉眼观察,梗死区变苍白为结扎成功的标志。缝合各层肌肉,缝皮,撤呼吸机。对照组只开胸不结扎。术后大鼠保温,单笼放置,待清醒后再次称重、编号,按组分笼饲养。以上手术均在无菌条件下进行,术后肌内注射青霉素钠2×105 U/d,连续5天抗感染。严格按照以上规程执行操作,以保证结扎部位、结扎操作时间和其他条件的一致性。心肌梗死+卡托普利组大鼠于术后将卡托普利(25mg/片,天津力生制药公司生产)按10mg/kg溶于水,立即肌肉注射。以后每日上午按卡托普利50mg/kg溶于水2ml灌胃。对照组和心肌梗死组均肌注和喂给等容量生理盐水。术后开始计时,于6时、12时、18时、24时、3天、5天、7天和14天(第3天到第14天抽血时间为每天上午8:00)分别抽大鼠静脉血1ml,放于试管中,以3000r/min离心10分钟,并立即用塑料吸管将上层血清转移至离心管中,标上组别、鼠号和时间,置于-80℃冰箱保存,于14天时集中测定BNP、ET-1和NO指标。大鼠血清BNP和ET-1浓度测定采用双抗夹心酶联免疫法,试剂购自于美国Rapidbio(RB)公司,酶标仪为河北医科大学第一医院检验科的型号为TECAN的仪器。大鼠血清NO浓度测定采用硝酸还原酶比色法,试剂购自于南京建成生物工程研究所,仪器为河北医科大学第一医院检验科的721分光光度计。所有数据用SPSS13.0 for windows软件包进行统计。大鼠血清BNP、ET-1和NO水平及动态变化用重复测量设计的方差分析,当总的组间变异F值有意义时,数据用SNK(Student-Newman-Keuls)检验以比较各组之间均数差异。为比较各具体时间点的组间差异,采用单因素方差分析,若F值有意义时,再用SNK检验。所有数据采用均数±标准误(mean±SEM)表示,检验水准а=0.05。结果:1大鼠血清BNP、ET-1和NO水平的组间比较:1.1 AMI组、AMI+capt组和对照组三组相比,各组间血清BNP和ET-1水平在每个时间点均有明显差异(各P值均<0.05),其有AMI组>AMI+capt组>对照组的大小关系。1.2 AMI组与对照组相比和AMI组与AMI+capt组相比,血清NO水平在每个时间点均有明显差异(各P值均<0.05),其有AMI组<对照组和AMI组<AMI+capt组的大小关系;AMI+capt组与对照组相比,血清NO水平在7天前的每个时间点均有明显差异(各P值均<0.05),有AMI+capt组<对照组的大小关系,第7天和第14天时两组水平无差异(P7d=0.164, P14d=0.678)。2各组大鼠血清BNP、ET-1和NO水平的动态变化:2.1大鼠血清BNP水平的动态变化AMI组大鼠血清BNP呈双峰分泌,第一峰峰值高于AMI+capt组,出现时间也早,而AMI+capt组血清BNP浓度只有一个峰值,对照组血清BNP浓度变化较平坦。(见Fig.1)2.2大鼠血清ET-1水平的动态变化AMI组和AMI+capt组大鼠血清ET-1动态变化均有一个峰值,但AMI组峰值高且出现早,对照组血清ET-1浓度变化较平坦。(见Fig.2)2.3大鼠血清NO水平的动态变化AMI组和AMI+capt组大鼠血清NO动态变化均有一个谷值(最低值),但AMI组谷值低且出现早,对照组血清NO浓度变化较平坦。(见Fig.3)结论:1 AMI组大鼠血清BNP和ET-1水平高于对照组,血清NO水平低于对照组,说明血清BNP、ET-1升高和NO降低参与了AMI后病情演变过程。2 AMI+capt组大鼠血清BNP和ET-1水平低于AMI组,血清NO水平高于AMI组,说明卡托普利能改善AMI后血清BNP、ET-1升高和NO降低程度,对AMI后病情变化有益。3大鼠AMI后血清BNP的浓度呈双峰分泌,在12.6±0.60小时达到第一峰值(412.00±3.95pg/ml) ,第二峰值(336. 58±1.54 pg/ml)出现在第153.60±7.33小时,此后继续下降,但在第14天时仍高于对照组。大鼠AMI后血清ET-1迅速升高,于7.8±0.92小时达一高峰(272.12±1.27pg/ml)后逐渐降低,在第14天时仍高于对照组。而血清NO迅速降低,于11.4±0.60小时达一低谷(16.98±0.35μmol/L)后逐渐升高,在第14天时仍低于对照组。4根据AMI+capt组大鼠的血清BNP、ET-1和NO峰值大小和出现时间,进一步说明卡托普利能抑制AMI后神经内分泌激活,使血清BNP、ET-1升高和NO降低程度改善,对AMI后病情大有裨益。更多还原

【Abstract】 Objective: Currently, coronary artery disease has become one of the main disease of harming the human’s health, especially acute myocardial infarction (AMI). After AMI occurs, larger area of myocardial infarction often leads to left ventricular systolic and diastolic dysfunction and evolves into congestive heart failure. Recent reseach found that the activation of neurohormonal system after AMI has a crucial effect on the evolvement of AMI into congestive heart failure. The moderate activation can mitigate the bad change of hemodynamics that occurs because of myocardial infarction and left ventricular systolic and diastolic dysfunction, so that the blood pressure becomes steady and the important apparatus are provided enough blood stream. However, the excessive activation leads to the apoptosis of cardiac muscle cells and the ventricle remodeling ,so that the excessive activation and the latter two progresses form a bad cycle that make the heat after AMI become congestive heart failure.Therefore, the reseach on the activation and the progress law of neurohormonal system after AMI have a significance on the diagnosis and treatment about the disease. As two important members of neurohor-monal system, BNP and endothelium function have important and particular action on Cardiovascular system. We designed this study to find the evolvements of brain natriuretic peptide(BNP), entothelin-1(ET-1) and nitrogen monoxide(NO) serum levels in rats after AMI and observe the effects of the captopril on the evolvements in order to offer new theories for the diagnosis and treatment about AMI.Methods: 30 male wistar rats weighting about 250g,about from 13 to 16 week-ages,were used in our study. Before operation the rats were randomly divided into three groups:①sham group (n=10) ;②AMI group (n=10) ;③AMI+capt group (AMI+captopril,n=10). Operation course: First injected 10% Chloral Hydrate to every rat’s abdominal cavity according to 35mg/kg.After narcosis they were fixed on an operation stage. Sent a thin tube into the glottis of trachea and connected it to a ventilator. It was a certificate to put the tube successfully that the rat’s stethidium rose and fell with the respiratory rate of the ventilator.Set the breath parameters.After careful disinfection to surgical incision with 75% alcohols, the heart was exposed through a 1.0~1.5cm left lateral thoracotomy. Broke the pericardium, extruded the heart quickly and exposed the left ventricular dissociative wall. After the left coronary artery was found lying between the left artial appendage and the pulmonary artery taper, the anterior desecending artery was ligated at about 2-3mm below the beginning part of left coronary artery with the 6-0 size silk thread. The ligation deep was 0.3-0.5mm. After the heart was laid back and the gas was drawn out, the thorax was closed.The ligation step needed about 30 second. It was thought of a successful ligation that the infracted part became white during operation. Oversewed all layers of muscle and skins and removed the ventilator. In the sham group rats, just a thoracotomy was made, a ligation was not. After operation the rats needed presevate them heat and be laid in a single cage. After regaining consciousness, the rats were again weighed in and numbered. Whereafter, thay were divided into three groups and laid in different cages and fed. The above operation must be carried through under the sistuation that have no germ. After operation the muscle injecting with ampicillin according to 2×105 U/d for 5 days prevented the infection. Strictly keeping to the protocol ensured the ligation part and time and other conditions consistentDissolve captopril in 1ml water according to 10mg per kilogram after operation.Then the rat was injected quickly. From next day on captopril (50 mg/kg) dissolved in 2ml water was given through gastric tube every morning, whereas the sham group was given equal physiological saline water.1-ml blood samples were obtained the sixth hour after operation;every 6 hours during the first 24 hours;thereafter once a day within the first week;and in the second week. Blood sampling from the third day through the second week was done at 8:00AM. Blood samples were taken from the rat’s vein and placed into tubes. A centrifugal machin was used to rotate at 3000r/min speed in 10 minute in order to separate the serum from the blood.The serum was transferred into centrifugal tubes that were adhibited tags with group numbers, rat numbers and times.Thereafter, the serum immediately was stored at -80℃until analysis.Rat’s serum concentrations of BNP and ET-1 both were measured with an enzyme-linked immunosorbent assay(ELISA). Reagents were purchased from American Rapidbio(RB) corporation.Their measurement instrument was the TECAN from the clinical laboratory of the first hospital of Hebei medical university. Rat’s serum concentrations of NO was measured with a nitrate reductase colorimetry. Reagents of NO were purchased from Nanjing Jiancheng Bioengineering Institute. It’s measurement instrument was the 721 spectrophotometer from the clinical laboratory of the first hospital of Hebei medical university.SPSS 13.0 software pack was used to make statistical analysis. The serum levels of BNP, ET-1 and NO were compared over the time course using ANOVA for repeated measures. Where the F value was found to be significant, the data were compared with Student-Newman-Keuls tests. One-Way ANOVA was used in order to compare the datas from sham group,AMI group and AMI+capt group at a specific time. Where the F value was found to be significant, the datas were compared with Student-Newman-Keuls tests. All values were expressed as mean±SEM. Statistical significance was defined as P<0.05.Results:1 Comparison About Serum Levels of BNP, ET-1 and NO in Rat From Sham Group,AMI Group and AMI+capt Group1.1 At every time, there all was a sginificant difference in the serum levels of BNP and ET-1 from AMI group,AMI+capt group and sham group.Compared with AMI group, the serum levels of BNP and ET-1 at every time in AMI+capt Group and sham group both was smaller(P<0.05).Similarly, compared with AMI+capt group, the serum levels of BNP and ET-1 at every time in sham group was also smaller(P<0.05).1.2 At every time, there all was a sginificant difference in the serum levels of NO between AMI group and AMI+capt group and between AMI group and sham group. Compared with AMI group, the serum levels of NO at every time in AMI+capt group and sham group both was bigger(P<0.05). At every time before the 7th day, there all was a sginificant difference in the serum levels of NO between AMI+capt group and sham group. Compared with AMI +captopril group, the serum levels of NO at every time in AMI +captopril group and sham group both was bigger till the 7th day(P<0.05).In the 7th day and the 14th day, there was no sginificant difference between AMI+capt group and sham group(P7d=0.164, P14d=0.678).2 Time Course of Serum Levels of BNP, ET-1 and NO in Rat From Sham Group,AMI Group and AMI+capt Group2.1 The serum BNP level in AMI group rat had two peak values. Whenas, the serum BNP level in AMI+capt group rat had one peak value. The first peak of the serum BNP level in AMI group rat was higher and earlier than that of AMI+capt group rat. The serum BNP level in sham group rat was smoother. (Fig.1)2.2 The serum ET-1 level in AMI group rat and AMI+capt rat both had one peak value. The peak of the serum ET-1 level in AMI group rat was higher and earlier than that of AMI+capt group rat. The serum ET-1 level in sham group rat was smoother. (Fig.2)2.3 The serum NO level in AMI group rat and AMI+capt rat both had one bottom value. The bottom of the serum NO level in AMI group rat was lower and earlier than that of AMI+capt group rat. The serum NO level in sham group rat was smoother. (Fig.3)Conclusions:1 Compared with sham group rat, the serum levels of BNP and ET-1 at every time in AMI group rat both was higher; by contraries, the serum levels of NO was lower. This showed that the increase of BNP and ET-1 serum levels and the decrease of NO serum level participated in the progress of AMI.2 Compared with AMI group rat, the serum levels of BNP and ET-1 at every time in AMI+capt group rat both was lower; by contraries, the serum levels of NO was higher. This showed that the captopril could lessen the increase of BNP and ET-1 serum levels and the decrease of NO serum level and was beneficial to the progress of AMI.3 In AMI group rat, the serum BNP level had two peaks: the first peak at 12.6±0.60 hours and the second peak at 153.60±7.33 hours after operation. Thereafter, the level decreased gradually but was still higher in the 14th day than that of sham group rat. The serum ET-1 level in AMI group rat increased after operation, forming a peak of 272.12±1.27pg/ml at 7.8±0.92 hours. Thereafter, the level decreased gradually but was still higher in the 14th day than that of sham group rat. The serum NO level in AMI group rat decreased after operation, forming a bottom of 16.98±0.35μmol/L at 11.4±0.60 hours. Thereafter, the level increased gradually but was still lower in the 14th day than that of sham group rat.4 Based on the peak value and the forming time of the peak of the serum BNP and ET-1 serum levels, and based on the bottom value and the forming time of the bottom of NO serum level in AMI+capt group rat, it was more shown that the captopril could restrain the activation of neurohormonal system and was better beneficial to the progress of AMI.更多还原