【作者】 刘忠保；

【导师】 赵荣瑞； 刘慧荣；

【作者基本信息】 山西医科大学 ， 生理学， 2005， 博士

【摘要】 背景与目的: 近年来的研究表明,肾素-血管紧张素系统(RAS)在心血管疾病的发生、发展中起着非常重要的作用。目前认为RAS 的主要效应分子血管紧张素系统II 有两种亚型受体,即AT1 和AT2 受体。研究表明血管紧张素II的生物效应主要是通过AT1 受体实现的。该受体不仅在调节心脏和血管活动中起重要的生理性调节作用,而且它的过度活动还能引发许多重要的病理生理后果,因而在诸多心血管疾病的发病中起着关键性作用。近年来,在许多心血管疾病患者的血清中发现存在有心脏G-蛋白偶联受体家族中几种主要受体(如肾上腺能β1 受体、α1 受体、胆碱能M2 受体等)的自身抗体,并证明这些自身抗体均能专一性识别相应受体的细胞外第二环功能表位肽段,并对各该受体表现有激动剂样的刺激效应,且抗体对受体的刺激作用与激动剂不同,没有脱敏现象,因而可呈现持续的刺激效应,而引发病理改变。这提示在心血管疾患的发病中有受体抗体的免疫学机制参与。最近几年,发现在先兆子痫和恶性高血压患者血清中存在有抗AT1受体自身抗体,但其参与发病的作用尚不清楚。为阐明与心血管疾病有重要关系的AT1 受体的自身抗体在心血管疾病发病中的作用,须首先了解AT1 受体抗体对心血管系统的生物效应,故本实验研究了AT1 受体抗体对心血管功能的影响及其机制,实验分以下五项内容: 一. 抗血管紧张素AT1受体抗体的制备 目的: 近年来的研究发现,许多心血管疾病患者的血清中存在有心脏G-蛋白偶联受体家族中几种主要受体的自身抗体,并证明这些自身抗体均能专一性识别相应受体的细胞外第二环功能表位肽段,且对各该受体表现有激动剂样的刺激效应。由于各受体的氨基酸序列在不同种属间有高度保守性,并证实心血管疾病患者血清中的抗受体自身抗体与用它们的受体靶肽段作为抗原免疫动物所生成的抗肽抗体具有相同的免疫与功能特性。因此我们利用人工合成的人AT1 受体细胞外第二环表位功能肽段,主动免疫大鼠,使其产生高滴度的抗AT1 受体抗肽抗体,借以进行抗体对心血管活动的生物学效应研究。材料和方法: 研究用的人AT1 受体细胞外第二环表位功能肽段(165-191 位)由华中科技大学同济医学院合成。其氨基酸序列为: I-H-R-N-V-F-F-I-E-N-T-N-I-T-V-C-A-F-H-Y-E-S-Q-N-S-T-L 使用上述的抗原肽段对Wistar 大鼠进行主动免疫,获取抗AT1受体抗血清,其效价用SA-ELISA 方法检测。然后利用MAb Trap Kit 试剂盒对抗血清中的抗体IgG 进行提取和纯化。纯化后的抗体蛋白液混合后,先经PAGE 胶凝胶电泳检测其纯度,然后使用考马斯亮兰试剂盒对抗体进行定量。结果: 1. 主动免疫的结果: 抗体滴度从免疫后的第二周开始升高(图1;表1)。第二次加强免更多还原

【Abstract】 Background and Aim: In recent years, it has been demonstrated that the renin-angiotensin system （RAS） plays a crucial role in the pathogenesis and development of cardiovascular diseases. At present it is commonly held that for angiotensin II, a dominant effective molecule of RAS, there are two receptor subtypes, i.e. AT₁ and AT2. The biological activities of angiotensin II are primarily mediated by angiotensin II AT₁ receptors. These receptors not only play an important role in the physiological regulation of cardiovascular activities but also their over-activity may lead to many pathophysiological consequences, and hence have a key role in the pathogenesis of various cardiovascular diseases. For the past few years, the existence of autoantibodies against cardiovascular G-protein coupled receptors including β₁-and α₁-adrenoceptors, M2-muscarinic receptors, etc, in the sera of patients with various cardiovascular diseases has been reported. These autoantibodies are able to specifically recognize the epitope of the second extracellular loop of the corresponding receptors and display a stimulatory “agonist-like”activities on their corresponding receptors. At variance with agonists, the antibodies stimulate the receptors without desensitization, and thus reveal permanent stimulatory effects resulting in pathological changes. This suggests the involvement of immunologic mechanism by receptor antibody in the pathogenesis of cardiovascular diseases. More recently, autoantibodies against angiotensin II AT₁ receptors have been found in the sera of patients with preeclampsia and malignant hypertension. However, the implicative role of these autoantibodies in the pathogenesis of the diseases are unclear. In order to clarify the role of antibody against functionally important angiotensin II AT₁ receptors in the pathogenesis of cardiovascular diseases, it is of significance to understand first the biological activities of these autoantibodies on cardiovascular system. Therefore, in this study we explored the effects of anti-AT₁-receptor antibodies on cardiovascular activities in rats. The present study consists of following five experiments. I. Preparation of anti-AT₁-receptor antibodies in rats Aim: To produce enough anti-AT₁-receptor antibodies （AT₁-R Ab） in the sera from rats immunized with antigenic peptide corresponding to the second extracellular loop of human angiotensin II AT₁ receptors. Methods: Peptide synthesis, Active immunization of rat model, detection of anti-AT₁-receptor antibodies by SA-ELISA assay, Affinity purification of IgG. Results: 1. Titres of AT₁-R Ab began to increase from second week after active immunization （Fig 1, Table 1）. At 8th week, the antibody titres in the sera of rats increased from the pre-immunization value of 1 : 25.56±9.22 to 1 :1490.00±847.58（p<0.01）. No changes were observed for control group.2.Purified immunoglobulin fractions （IgG） from the positive sera were prepared using a MabTrap kit （Amersham）. The concentration of IgG after purification is 2.38 mg/ml.These purified AT₁-R Ab IgG were then used in the following studies on the biological activities of AT₁-R Ab. II. Effects of AT₁-R Ab on rat ventricular myocyte ionic currents Aim: In an attempt to clarify the role of AT₁-R Ab in the pathogenesis of cardiovascular diseases, the direct effects of AT₁-R Ab on the cardiac performance should be first investigated. In view of the myocardial electrical activities are the fundamental bases of various cardiac performances and that the research at cellular level is very suitable for the study of the direct action of antibody, so in this study we observed first the effects of AT₁-R Ab on some ventricular ionic currents. Methds: Whole cell patch clamp techniques were used to study the effects of AT₁-R Ab on rat ventricular I_Ca-L, I_Na/Ca, I_to, and I_k1 currents which were compared with those of Ang-II. The actions of Losartan, a specific antagonist of AT₁-receptor, on these effects were also analyzed. Results: 1.Effects on ICa-更多还原