慢性复合应激诱发高血压动物模型的制备钟航海1 冯英凯2作者简介：钟航海，男，主治医师主要从事心血管疾病发病机制及诊治研究通讯作者：冯英凯（1967~），男，博士后，副主任医师Email：fykme@(1.重庆市九龙坡区第五人民医院内科 401329；2. 第三军医大学西南医院病理学研究所)【摘要】 目的 应用多重刺激手段，模拟上班白领族遭受的常见复合应激因素，制备慢性应激性高血压大鼠模型，为进一步探讨SIH的发生、发展机制奠定基础方法 健康成年Wistar大鼠30只随机分为传统模型组（n=10）、自建模型组（n=10）及正常对照组（n=10）传统模型组接受足底电击结合工业噪声复合刺激，自建模型组应用不规则震荡、不良音乐、部分睡眠剥夺复合刺激法正常对照组不接受应激刺激建模时间为4周，在应激刺激第0、7、14、21、28日（分别以D0、D1、D7、D14、D21、D28表示）测定各组收缩压及心率第29日颈动脉插管取血4 mL，分离血浆，酶联免疫吸附法（ELISA）法检测血浆血管紧张素Ⅱ（AngⅡII）及皮质醇水平，高效液相色谱（HPLC）法测定血浆儿茶酚胺含量结果 传统模型组收缩压在应激刺激第7天较基础值上升约15 mmHg，在第14天达高峰[（140.6±4.3）mmHg]，而后略下降，但仍维持在高水平直至第28天；自制模型组SBP变化趋势与传统模型组类似，但上升时间及达高峰时间均晚1周；在观察期，正常对照组收缩压平稳。
传统模型组及自制模型组心率在应激刺激1周后即开始增加，自第2周开始维持心率在较高水平，与正常对照组比较，差异有统计学意义（P<0.05）传统模型组及自制模型组血浆血管紧张素ⅡII、去甲肾上腺素、肾上腺素、皮质醇水平均显著高于正常对照组，差异均有统计学意义（P<0.05）结论 应用不规则震荡、不良音乐、部分睡眠剥夺复合刺激法可成功诱发实验大鼠产生应激性高血压，成功制备应激性高血压大鼠模型关键词】应激性高血压；动物模型；酶联免疫吸附试验；高效液相色谱Preparation Establishement of chronic combined stimuli-induced hypertension animal model ZHONG Hang-hai1, FENG Ying-kai2. 1. Department of Internal Medicine, the Fifth People’s Hospital of Jiulongpo District of Chongqing City, Chongqing 400029, China; 2. Institute of Pathology, Westsouth Hospital of Third Military Medical University, Chongqing 400038, China【Abstract】 Objective Multipe stimuli were adopted to simulate the common combined stressors from which the white-collar class suffers. The aim of the study is to develop chroinc sreess induced hypertension (SIH) rat model, so as to settle the basis for further investigation on occurrence and development of SIH. Methods Totally 30 healthy adult Wistar rats were randomly divided into traditional model group (n=10, receiving combined stimulation of electric foot shock and industrial noise), self-made model group (n=10, receiving combined stimulation of irregular shake, infaust music and partial sleep deprivation) and normal control group (n=10, no stimuli given). The modeling duration was 4 weeks. Systolic blood pressure (SBP) and heart rate (HR) were monitored at different time points (0, 7, 14, 21 and 28 days after first stimuation, which were denoted as D0, D1, D7, D14, D21 and D28 ) . On day 29, cervical arterial cannula was carried out to obtain the whole blood 4 mL; then the plasma was isolated. Enzyme linked immunosorbent assay (ELISA) was applied to determining plasma levels of angiotensin Ⅱ (AngⅡ) and cortisol, and high performance liquid chromatogram (HPLC) was adopted to measure the content of plasma catecholamine. Results In traditional model group, SBP began to rise at D7 time pint, about 15 mmHg above the basic value, peaking at D14 time pint (the highest SBP was （140.6±4.3）mmHg ) , and declining afterwards, but still keeping a higher SBP level at D28 time point. The changing tendency of SBP in self-made model group was similar to that in traditional model group, notwithstanding the initial rising time and peaking time were one week later. In the viewing period, SBP kept stable in normal contrl group. Heart rate initiated to rise at D7, and from the second week on, HR kept at a higher level in traditional model group and self-made model group, significantly higher than that in normal contrl group (P<0.05). The plasma levels of AngⅡ, norepinephrine (NE), epinephrin (E) and cortisol were markedly higher traditional model group and self-made model group than those in normal contrl group. The differences were statistically significant (P<0.05). Conclusion Combined stimuli of irregular shake, infaust music and partial sleep deprivation succssfully provokes the experimental rats’ stress-induced hypertension. Self-made SIH rat model is finally developed. 【Key words】 sreess induced hypertension (SIH); animal model; Enzyme linked immunosorbent assay (ELISA); high performance liquid chromatogram (HPLC) 随着现代科技进步和社会经济的迅猛发展，生活节奏不断加快，应激性高血压（stress-induced hypertention，SIH）的发病率逐年升高，已成为危害人类健康，特别是上班白领族的隐形“杀手”。
本组应用多重刺激手段，模拟上班白领族遭受的常见复合应激因素，制备慢性应激性高血压大鼠模型，并与足底电击结合噪声复合刺激制备的传统模型进行比较，为进一步探讨SIH的发生、发展机制，研究更加有效的预防治疗措施奠定基础未涉及研究背景和SIH的研究进展，建议补充另外，集中复合刺激因素如电、噪音、震动、睡眠剥夺这些处理因素选择的依据何在？和文献报道有何异同？均需要在文中有所描述1 材料与方法1.1 动物的选择与分组 以首次给予应激刺激时动物表现迅速逃避、尖叫、竖尾、喘息并不易适应为标准，选择健康成年清洁级Wistar大鼠30只，雌雄不拘，体质量200~250 g，由第三军医大学实验动物中心提供饲养温度为23~25℃，湿度为40%~45%，混合饲料喂养利用随机数表将30只大鼠随机分为自建模型组（n=10）、传统模型组（n=10）及正常对照组（n=10）1.2 仪器与试剂 MG-2型迷宫刺激器，由江苏省张家港市生物医学仪器厂生产；RBP-1B型大鼠血压心率测定仪，由中日友好临床医学研究所研制；血管紧张素Ⅱ（angiotensin Ⅱ，AngⅡ）酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）试剂盒购自上海工硕生物技术有限公司提供公司，兔抗大鼠AngⅡII抗体购自武汉博士德公司；Agilent 1100高效液相色谱仪为美国安捷伦公司产品；大鼠皮质醇（cortisol）ELISA试剂盒购自武汉优尔生科技股份有限公司（Uscn Life Science Inc.）。
去甲肾上腺素（norepinephrine，NE）标准品（023K0956）及肾上腺素（epinephrine，E）标准品（023K0955）均为美国Sigma公司产品1.2 模型制备 传统模型组：参照董艳芬等[1]方法，将大鼠分别置于MG-2型迷宫刺激器的分格箱中，每天上午8:00-10:00、下午14:00-16:00用低频、低压交流电电击大鼠足底并结合100 dB工业噪音同步刺激，连续4周自建模型组：应用不规则震荡、不良音乐、部分睡眠剥夺复合刺激法震荡在文献[2]基础上作适当改进上午8:00将大鼠放入制动器中，放在DS-2水平摇床上进行震荡100次/min震荡45min→120次/min震荡60 min→100次/min震荡 45min，震荡期间间断关闭水平摇床电源不良音乐：下午14:00-17:00循环播发一些压抑、恐怖的背景音乐如《嫁衣》、《幸福大街》、《黑色星期五》大鼠对压抑恐怖音乐的感受是否和人类。