摘要
前言
冠心病是严重威胁人类生命健康及生存质量的疾病,为中老年人三大主要死因之一。冠状动脉血运中断后,该血管支配区(危险区)内的心肌细胞缺血、缺氧,并逐渐发生细胞坏死。近年来,就如何最大限度地发挥与利用机体自身的抗损伤机制,各国学者进行了大量而深入的研究,心肌缺血预适应(ischemic preconditioning,IPC)就是最受瞩目的热点。
IPC定义为:反复短暂的冠脉缺血可以对抗随后长时间的冠脉缺血,产生减少梗塞面积的心脏保护作用。预适应保护存在两个时间段,第一个是在预刺激后即刻产生,并持续1~3小时的早期预适应,也被称为经典预适应,也是本研究所涉及的时程。另外一个阶段是延迟期预适应,它是在最初预刺激损伤出现后的12~72小时内发生的心肌保护现象,被命名为第二窗保护(SWOP)。
心肌缺血预适应对心肌的保护是迄今为止发现的最为强大的内源性保护,因此探讨IPC的特点及机制并服务于临床是目前各国学者研究的热点。然而IPC激发心肌抗缺血保护的确切机制至今未明。本课题以深入研究心肌缺血预适应的机制为目的,以目前国内外研究较少的β-肾上腺素受体信号转导通路、一氧化氮—环磷酸鸟苷信号通路及蛋白酪氨酸激酶活性变化为主线,旨在深入研究几条不同的信号转导通路在早期心肌缺血预适应中的作用机制及意义,以更好地为临床服务。
实验材料
一.实验动物
健康SD大鼠,体重270~320g,由中国医科大学实验动物部提供。
二.实验试剂
TTC,Evan's Blue,心肌酶谱试剂盒,~3H—双氢心得舒,cAMP放免分析试剂盒,[γ-~(32)P]ATP,蛋白激酶A(PKA)单克隆抗体(第一部分);一氧化氮试剂盒,一氧化氮合酶(NOS)活性测定试剂盒,cGMP放免测定试剂盒,内皮型一氧化氮合酶(eNOS)抗体,免疫组织化学试剂盒(第二部分);[γ
/中二ATP,谷蛋白一酪蛋白聚合物,Whatman PSI离子交换滤纸(第三部
分)。
三.实验仪器
动物人工呼吸机,心电图机,低温高速离心机,显微图像分析系统,液问
计数仪,电泳槽,72分光光度计,石蜡包埋全自动处理仪,恒冷箱冰冻切片
机。
实验方法
各部分首先应用套管法制作在体大鼠心肌缺血/再灌注损伤模型:大鼠
经3%异戊巴比妥钠N0mg/Kg)腹腔注射麻醉后,气管插管,呼吸机辅助呼
吸,接心电图肢体导联。沿胸骨左缘 0.5 cm剪断第 23上肋骨,以手术线牵
拉两侧肋骨暴露胸腔。破心包,以肺动脉圆锥与左心耳之间的左冠状静脉
为标志,在左冠状静脉下缝线,线两端共穿过一根聚乙烯小管以形成闭环。
拉紧闭环并用止血钳固定即产生缺血,放松闭环即发生再灌注。实验中以
左室前壁紫组及同步心电图标11导联S-T段抬高为结扎成功标志,对变
化不明显者、穿线中大量出血者剔除实验。
第一部分
制作大鼠在体心肌缺血/再灌注损伤模型,将动物分为预适应组一P
组,n=12人手术穿线后平衡 sndn后结扎 smin,放松 smin,反复 3次后,持
续结扎30min,再灌注 90min,结束实验。缺血/再灌注组 VR组,n二 12人
手术穿线平衡35ndn后,持续结扎30ndn,再灌注90min,结束实验。假手术
组*ON组,n=6入手术穿线后,不予处置,155ndn后结束实验。预适应程
序组*C组,n=36),在 PC组中进一步分为N-,2-,3-,和 PCI+,
2+,3十组,每组n=6。其中3周期sndn 缺血示为PCI-,2-,3-;间隔的
smin再灌注示为 PCI\2\3+。实验程序结束后,取血测量心肌酶谱*P
组与 I/R组各取6只行EvanA Blue及TI’C染色后,用计算机图像分析系统
比较危险区\梗死区面积及与左室总截面积的百分比关系。其余心脏匀浆
后应用差异离心法分离胞浆与胞膜,o仰O法测定蛋白含量。胞膜应用放
射受体分析法,Scatchnd作图测量p一肾上腺素受体(p-AR)最大结合力
问max)及密度(KD人胞浆应用放射免疫法测量cAMP含量,生化法测量
PKA/C活性,免疫引迹分析测量PKA含量。
·2·
第二部分
模型制作及分组同第一部分。IP组及I/R组各取6只大鼠于实验程
序结束后,取心脏平行房室沟将左室切成*5-Zrnm薄片共5片,然后进行
石蜡包埋,以SABC法进行内皮型一氧化氮合酶*NOS)免疫组织化学研
究。其余大鼠于实验程序结束后,取心匀浆后离心制备胞浆,o W’一法测定
蛋白含量。放射免疫分析法测定。GMP含量,生化法应用药盒测定NO含
量、NOS活性变化。
第三部分
模型制作及分组同第一部分。于实验程序结束取心匀浆后,离心制备
胞浆,o Wr法测定蛋白含量,生化法测定o?:K活性。
实验结果
第一部分
门X心肌酶谱检测CK人K——MB及LDH,三种酶在IP组较I/R组有显
著降低,P<o.01。IP组与*ON组之间,*K人K*B有显著差异人*H未
见明显差异。
p)IP组与I/R组比较左室总截面积,危险区面积,危险区/左室总截
面积,差别不显著。但I/R组梗死区总面积经图像分析软件分析定量为
7037 223437?
Preface
Coronary artery disease is one of the three major diseases which threaten the health status of the elderly. Once the coronary blood flow stopped, ischemia and anoxia will happen in the myocardial cells of that area. In the recent years, many studies have been developed in the field of how to use the anti - injury mechanism of organism itself. Much attention have been focused upon ischemic preconditioning (IPC).
Repeated episodes of ischemia and reperfusion dramatically limit infarct size of myocardium caused by subsequent prolonged period of ischemia. This phenomenon is called " ischemic preconditioning". There are two phases of IPC. One is induced immediately after pretreatment and last about one to three hours, which is called " classic ischemic preconditioning". The other is delayed phase which is induced about twelve to seventy - two hours after pretreatment and is called " second window of protection ( SWOP ) " .
IPC has come to be acknowledged as the most consistently powerful and reproducible method of delayed the development of ischemic injury known to date. Therefore, it is a hot - spot to study the mechanism of IPC and use them to serve clinical research. However, the accurate mechanism of IPC which induce anti -injury of ischemia has not been concluded till now. As knowledge of IPC accumulated, it became apparent that several signal transduction pathways may participate.
In this paper, we aim to further study the mechanism of IPC and research three signal transduction pathways of IPC, they are the b-adrenergic signal transduction, nitric oxide ( NO) - cGMP signal transduction and protein tyrosine kinase(PTK) pathways. As we know to date, there are only a few studies have
concentrated these three pathways.
Materials
1. Animals: Female SD rats ( supplied by Animal Laboratory Center of China Medical University) weighing 270 to 320g were used.
2. Drugs: Triphenyltetrazolium chloride (TTC) , 3H - DMA, cAMP RIA kit, [r-32P]ATP, PKA antibody (the first part) ; NO kit, NOS kit, cGMP RIA kit , eNOS immunohistochemistry kit ( the second part) ; [r-32P] ATP, gluten - casein polymer, Whatman P81 ion - exchange filter paper ( the third part).
3. Equipment: Animal respirator, Nihon Konden electrocardiograph, stereoscopic microscope, liquid scintillation counter, electrophoresis bath, 721 spectropho to meter, paraffin imbedding full - automatic apparatus, microtome.
Methods
In every part, a rat model of myocardial ischemia/reperfusion injury in vivo was used for these studies. Rats were anesthetized with 3% sodium pentobarbital (60mg/Kg) , intubated, and mechanically ventilated with a respirator with room air. ECG was monitored by bipolar limb leads. Left anterolateral thoracotomy was performed from the second to the fourth intercostals space, and the pericardium was opened. A 3 - 0 silk thread was then passed around the proximal of the left major coronary artery with a small curved needle, and its ends were threaded through a small polyethylene tube. Rats were allowed 5 minutes after surgical preparation to reach a steady state. Coronary occlusion was produced by pulling the snare and clamping it with a mosquito hemostat. Reperfusion was produced by releasing the clamp. Myocardial ischemia was confirmed by ST segment elevation of the ECG as well as observation of regional cyanosis over the myocardial surface.
Part One
The rats were divided into four groups: Ischemic preconditioning group (IP
group, n = 12) , rats were subjected three cycles of five minutes of ischemia followed by five minutes of reperfusion and then subjected to 30 minutes of ischemia followed by 90 minutes of reperfusion. Ischemia/reperfusion group (I/R group, n = 12) , After surgery, rats were balanced for 35 minutes and then subjected to 30 minutes of ischemia followed by 90 minutes of reperfusion. Control group (CON group, n =6) : After surgery, no procedures were made. After 155 minutes finish the experiment. Preconditioning procedure group ( PC group, n = 36): In this group, rats w
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