【作者】 李江；

【导师】 王莉；

【作者基本信息】 四川大学 ， 药理学， 2007， 博士

【摘要】 背景：充血性心力衰竭（congestive heart failure，CHF）是各种心脏疾病导致心功能不全的一种综合症，主要是指心肌收缩力下降使心排血量不能满足机体代谢的需要，器官、组织血液灌注不足，同时出现肺循环和（或）体循环淤血的表现。心力衰竭是多种心脏疾病的终末期表现，死亡率较高，是全世界关注的健康问题。寻找能够改善心力衰竭症状、血流动力学状况和预后的新疗法新药物是临床医生的愿望也是新药开发者的追求目标。Neuregulins是一种血管内皮来源的上皮生长因子，是酪氨酸激酶受体家族（ErbB）的激动剂。国内外研究表明，Neuregulins-ErbB信号系统在心脏发育过程和心肌对病理生理负荷或损伤的适应机制中都发挥重要作用，在心力衰竭中可能也起重要作用。外源性Neuregulins的生物治疗方法已经在大鼠模型中观察到了明显的改善作用，研究Neuregulins在灵长类动物心力衰竭模型中的治疗作用，探索其可能的作用机制对深入研究心力衰竭的分子发病机理和开发新型的心力衰竭治疗药物都有重要意义。目的：研究ErbB受体与蛋白激酶B在心力衰竭发生机制中的作用。研究重组人Neureguline对快速起博所致心力衰竭恒河猴心肌收缩能力的影响，探讨Neuregulin对快速起搏所致猴心衰心肌细胞损伤的保护作用及其可能的机制。方法：成年雄性恒河猴24只，随机分为假手术组、心力衰竭组及Neureguline治疗组，每组8只。治疗组240次／min起博7day后，起博状态下连续10d，每d 1次静脉注射给予重组人Neureguline 3μg／kg，心力衰竭组给予同体积生理盐水。连续给药10d结束后，测定左室内压最大上升速率（Lvdp／dtmax）、左心室舒张末期压（LVEDP）、左心室收缩末期压（LVSP）等血流动力学指标；测定射血分数（EF），每搏输出量（SV）、室间隔搏动幅度（ⅣS）等超声心动图指标；采用电化学发光免疫测定法观察脑钠肽（BNP）含量；通过TUNEL法检测心肌细胞凋亡指数；采用实时荧光定量PCR的方法分析肌球蛋白重链（α-MyHC）的表达水平；采用RT-PCR方法测定ErbB₂、蛋白激酶B（PKB）、Bcl-xl、Glut1 mRNA表达水平；采用Western Blotting法检测蛋白激酶B活性（phospho-PKB，即磷酸化蛋白激酶B蛋白水平）及凋亡相关基因Bcl-xl蛋白水平。结果：1快速起搏诱导心力衰竭模型恒河猴心肌收缩能力显著下降，心衰标志物脑钠肽水平明显上升（p＜0.05）。与对照组比较ErbB₂、PKB、Bcl-xl、Glut1 mRNA表达水平明显下降（p＜0.05），同时心肌组织蛋白激酶B活性显著下降，Bcl-xl蛋白水平也明显下降（p＜0.05）。2起搏状态下连续10天静脉注射给予3μg·kg^-1重组人Neuregulin后，左室内压最大上升速率（Lvdp／dtmax）显著升高，心衰标志物脑钠肽水平明显降低。3起搏状态下连续10天静脉注射给予3μg·kg^-1重组人Neuregulin后，α-MyHC表达水平明显上调。4起搏状态下连续10天静脉注射给予3μg·kg^-1重组人Neuregulin后，快速起搏诱导的心肌细胞凋亡受到明显抑制；同时心肌组织蛋白激酶B活性显著增强，Bcl-xl蛋白水平也明显增加。5起搏状态下连续10天静脉注射给予3μg·kg^-1重组人Neuregulin后，心肌组织蛋白激酶BmRNA表达及活性（磷酸化水平活性）显著增强，葡萄糖转运蛋白1（Glut1）mRNA水平明显上调。结论：1快速起搏所致猴心衰心肌细胞损伤的机制可能与ErbB₂，下游蛋白激酶B、凋亡抑制基因Bcl-xl、葡萄糖转运蛋白1（Glut1）表达水平下降有关。2静脉注射给予3μg·kg^-1重组人Neuregulin对快速起搏所致猴心衰心力衰竭有确切的治疗作用。3重组人Neuregulin可能通过细胞膜上的酪氨酸激酶受体ErbB结合，向上调节α-MyHC表达水平这一机制来增强心肌收缩力，从而达到治疗心衰的目的4 Neuregulin保护快速起搏所致猴心衰心肌细胞损伤的机制可能与蛋白激酶B的活性增加和凋亡抑制基因Bcl-xl蛋白水平上升有关。5重组人Neuregulin可能通过细胞膜上的酪氨酸激酶受体ErbB结合，向上调节Glut1表达水平，改善缺血心肌能量供应。更多还原

【Abstract】 Background Congestive hear failure （CHF） is a cardia insufficiency syndrome, caused by diverse cardiac diseases. The main mechanism is that the reduced contractile function of cardiac muscle can’t provide enough blood flow to surpport the metabolism of whole body. So the main symptoms of CHF are due to insufficient blood flow in organs and tissues, and present severe congestion in pulmonary circulation and systemic circulation. CHF is the final symptom of many cardiac diseases, and attracts worldwide attention for its high mortality. Clinicians have taken efforts to discover new therapy and new medicine to improve the cardiac function and hypoxia situation of patients, which is also the ideal aim of pharmaceutical researchers. Neuregulins is a endothelia derived epidermal growth factor, a excitomotor of tyrosine protein kinase receptor family （ErBb）. Many investigations have shown Neuregulins-ErbB signal system plays important roles in cardiac development and adaption of myocardium to pathological and physiological load and injure, and also is significant in CHF pathogenesis. Treatment of exogenous Neuregulins has been proved to be efficient way to improve CHF in rat model. So, carrying on researchs on the therapeutic effect of Neuregulins in primate model and investigating the possible mechanism will gain light on the molecular pathogenesis of CHF and benefit the exploitation novel drug for CHF.Objective To investigate the roles of neuregulin receptor ErbB₂ and protein kinase B （PKB） in Pacing-induced heart failure of rhesus monkey, to evaluate the effects of recombined human Neuregulin on contractibility of cardiac muscle in Pacing-induced heart failure of Rhesus Monkey and to clarify the possible mechanisms therein involved.Methods Twenty four rhesus monkeys were randomly divided into three groups （shame operated group,heart failure group and Neuregulin treated group）, each with 8 monkeys. Heart failure were induced by rapid pacing（240 heartbeats/min）. Neuregulin treated group received Neuregulin 3μg/kg intravenously for 10 days. Hemodynamic measurements such as the peak positive rate of change in left ventricular blood pressure （+ dP/dtmax） and Left ventricular systolic, end-diastolic blood pressures （LVSP and LVEDP, respectively） were performed. Ejection fraction was measured by echocardiogram. N-Terminal Pro-Brain Natriuretic Peptide （BNP）, one of the most important molecular marker of heart failure, was also measured by the method of electrochemical luminescence immunoassay. The apoptosis of cardiac myocyte was observed by Tunel method. Using real-time quantitative RT-PCR was used to detect the expression of myosin heavy chain mRNA in left ventricular cardiac muscle.The mRNA expression of ErbB₂, PKB, Bcl-xl and Glut1 were detected in the left ventricular free walls by RT-PCR method. The expression of phospho-PKB and Bcl-xl on protein level was also detected by Western blotting.Results The contractibility of cardiac muscle was significantly decreased, which consisted with the increase of BNP after rapid pacing. Increase of the collagen fibers were found in heart failure group. Rapid pacing was shown to induce cardiac myocyte apoptosis in rhesus monkey. The mRNA expression of a-myosin heavy chain, ErbB2, PKB, Bcl-xl and Glut1 were depressed in heart failure group, and the similar results was also found in the protein expression analysis of phospho-PKB and Bcl-xl. However the contractibility of cardiac muscle was significantly increased with the decrease of BNP treated with 3μg·Kg^-1 Neuregulin. In addition, the levels of mean expression of a-myosin heavy chain and Glut1 mRNA in Neuregulin treated group were significantly higher than that in heart failure. The apoptotic index was obviously lower in Neuregulin treated group. It also showed increase of Bcl-xl and more activity of PKB in those animals treated with 3μg.Kg^-1 Neuregulin.Conelution1 The expression of ErbB2, PKB, Bcl-xl and Glut1 were down-regulated during heart failure in rhesus monkey which suggested the important roles of ErbB₂ receptor and PKB in the mechanism of heart failure. 2 Recombined human Neuregulin can enhance the contractibility of cardiac muscle, relieve the heart failure syndrome.3 Through reversing the falling of a-myosin heavy chain induced by rapid ventricular pacing, recombined human Neuregulin can enhance the contractibility of cardiac muscle.4 Neuregulin protects against pacing-induced apoptosis in heart failure of rhesus monkey and the mechanism might be attributed to its increase of the activity of PKB and Bcl-xl protein.5 The improved effect of neuregulin on cardiac function might be attributed to the increase of the glut1 expression.更多还原