Cardiac function of myocardial infarction rat models and NSF-siRNA , a key protein released from vesoactive substance

BACKGROUND: How to reduce the incidence and mortality of cardiovascular diseases is an urgent concern in the field of public health. OBJECTIVE: To explore the influence of adenovirus-mediated NSF-siRNA release from vesoactive substance on the cardiac function of a rat model of myocardial infarction. METHODS: A total of 36 adult Sprague-Dawley rats were applied to establish acute myocardial infarction models by ligating the anterior descending branch of the left coronary artery. After the model was determined by electrocardiogram successfully, NSF-siRNA adenovirus (experimental group), negative adenovirus (control group) and normal saline (normal saline group) were injected near the infarct area of the left ventricle of rats respectively. After 2 weeks, the left ventricular ejection fraction (LVEF) was tested with noninvasive ultrasonic cardiogram. Meanwhile, the left ventricular end-diastolic pressure (LVEDP) and maximum pressure rising speed of left ventricular (dp/dt max) were detected by connecting the right external carotid artery place pipe to the BL-420 biological function experiment system, to evaluate the cardiac function. Subsequently, the rat heart was harvested for serial sections to observe the infarcts range. RESULTS AND CONCLUSION: After 2 weeks, the LVEF of the experimental group was increased remarkably (P < 0.05), while the LVEDP of the experimental group was decreased evidently compared with the control group and normal saline group (P < 0.05), and the dp/dt max of the experimental group was significantly increased (P < 刘岩,等. 心肌梗死模型大鼠心功能与血管活性物质释放关键蛋白 NSF-siRNA P.O. Box 10002, Shenyang 110180 www.CRTER.org 4288 www.CRTER.org 0.05). Furthermore, there were no significant differences in the infarct area among groups (P > 0.05). The local injection of adenovirus mediated NSF-siRNA expression vector in infarct part can improve the cardiac function indexes, including LEVF, LVEDP and dp/dt max 2 weeks after myocardial infarction, but it has no impact on the myocardial infarction area. Subject headings: myocardial infarction; models, animal; stroke volume; RNA interference Liu Y, Zhou Y, Yang SX, Wang Z. Cardiac function of myocardial infarction rat models and NSF-siRNA, a key protein released from vesoactive substance. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(27):4287-4292. 0 引言 Introduction N-已基顺丁烯二酰亚胺敏感因子(N-ethylamaleimidesensitive factor,NSF)是介导内皮细胞中可分泌颗粒韦伯 潘力氏小体(Weibel Palade body,WPB)与内皮细胞膜融 合而导致胞吐作用的关键蛋白质,介导释放韦伯潘力氏小 体内容物。韦伯潘力氏小体内含有P-选择素、血管性血 友病因子、内皮素1及白细胞介素8等血管活性物质,这些 物质在血管损伤的早期即被释放,可以导致中性白细胞黏 附到血管内皮、血小板聚集,并诱发血管痉挛等一系列血 管壁的炎症反应,在血栓形成、炎症反应和血管痉挛方面 起着至关重要的作用,与动脉粥样硬化、急性冠脉综合征 和心肌梗死等的发生发展相关密切。 课题组一直注重韦伯潘力氏小体释放的机制和调节释 放方面的研究,前期已经成功克隆出人主动脉内皮细胞 NSF基因,构建了针对人主动脉内皮细胞NSF蛋白N端功 能区编码基因的重组腺病毒载体Ad-H1-NSF/siRNA,通过 DNA测序及病毒滴度测定后转染于人主动脉内皮细胞,用 Realtime PCR及Western Blot技术在不同的时间点对 NSF-mRNA及NSF蛋白的表达进行测定,结果显示 NSF-siRNA能够诱导NSF-mRNA表达下调,降低NSF蛋白 合成,从而有效抑制内皮细胞韦伯潘力氏小体的释放。 本实验在前期研究的基础上,建立大鼠心肌梗死模型,在 梗死区周围局部注射重组腺病毒载体NSF-siRNA,应用心 脏超声和生物机能实验系统测定2周后大鼠心功能,以探索 NSF-siRNA对模型大鼠心肌梗死后心功能的影响,为开发 小分子RNA药物及防治动脉粥样硬化、急性冠脉综合征和 急性心肌梗死提供有价值的资料。 1 材料和方法 Materials and methods 设计:随机对照动物实验。 时间及地点:实验于2011年12月至2013年5月在首都 医科大学附属北京世纪坛医院动物实验室完成。 材料: 实验动物:SPF级SD大鼠36只,雄性,体质量为 180-220 g,购自北京维通利华实验动物技术有限公司,合 格证号为11400700010718,实验动物使用许可证号为 SCXK(京)2012-0001。动物在屏障系统环境下,用专用饲 料(购自北京维通利华实验动物技术有限公司)和灭菌水分笼 饲养。 实验方法: Adv-NSF-shRNA的构建和制备:从GeneBank中选取 大鼠NSF基因序列(基因收录号:NM_021748.1),采用 siRNA Target Designer-Version1.51设计软件,设计针对 NSF基因N-末端区的21 nt寡核苷酸干扰序列 (5’-TAG GAC TGG TCG TTG GAA ACA-3’),并设计siRNA正义链 和反义链,以loop(10 nt)相连,称为shRNA(short hairpin RNA)(图1)。合成编码shRNA的DNA模板的两条单链,退 火DNA单链得到shRNA的DNA双链模板。模板链后面连接 RNA Poly III聚合酶转录中止位点,同时两端分别设计 MluI和Hind III酶切位点,可以克隆到siRNA 载体腺病毒 SD1219多克隆位点的MluI和Hind III酶切位点之间(图 2)。腺病毒载体pRNAT-H1.1/Adeno(SD1219)经MluI和 Hind III双酶切后,1%琼脂糖凝胶电泳,回收线性载体。 与新合成的两端分别带有MluI和Hind III酶切位点 NSF-shRNA DNA模板使用T4连接酶进行连接。连接产物转 化DH5α感受态细菌,在LB-kana培养基上铺板接种,37 °C 倒置培养过夜。收获细菌,使用上海Shinegene公司的小量 质粒抽提试剂盒提取质粒DNA,并使用MluI和Hind III双 酶切鉴定和测序鉴定后标记冻存。Adv-NSF-shRNA的纯化 及鉴定参照文献 [7] 。将透析纯化后的腺病毒采用 TCID50(50% tissue culture infective dose)法进行病毒滴度 测定,计算出本次病毒制备的滴度为1.2×10 TU/L (Adv-NSF-shRNA由上海Shinegene公司构建和制备)。 心肌梗死模型建立:参考以往的模型制作方法,结扎 冠状动脉前降支制造心肌梗死模型。将36只SD大鼠称质 量,根据体质量将实验鼠排序,随机分为实验组(注射重组 腺病毒)、对照组(注射阴性腺病毒)、生理盐水组,每组12 NSF-siRNA干预后心肌梗死大鼠模型心功能检测所需主要仪器和试剂: