早期心肌缺血预适应信号转导机制的研究
|
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
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
引文
1 Murry CE, Jenning RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardiaum. Circulation 1986; 74:1124～1136
2 Kaszala K, Vegh A, Papp JG, et al. Time course of the protection against ischemia and repeffusion - induced ventricular arrhythmias resulting from brief period of cardiac pacing. J Mol Cell Cardiol 1996; 28:2085～2095
3 Mullane K, Bullough D. Harnessing an endogenous cardioprotective mechanism: sources and sites of action of adenosine. J Mol Cell Cardiol 1995; 27: 1041～1054
4 Wang G - Y, Wu S, Pei J - M, et al. K but not δ - opioid receptors mediate effects of isehemic preconditioning on both infarct and arrhythmia in rats. Am J Physiol Heart Circ Physiol 2001; 280: H384～H391
5 Lan X - L, Diao Y, Lan J - Ch, et al. The effect of aclrenergie receptor-adenyl cyclase system on myocardial ischemie preconditioning in rats. Nuclear Science and Techniques 2002; 13(4): 230～236
6 Schott RJ, Rohmann S, Braun ER, et al. Isehemie preconditioning reduces infarct size in swine myocardium. Circ Res 1990; 66:1133～1142
7 Morris SD, Yellon DM. Angiotension -converting enzyme inhibitors potentiate preconditioning through bradykinin B2 receptor activation in human heart. JACC 1997; 29:1599～1606
8 Ikonomidis JS, Tumiati LC, Weisel RD, et al. Preconditioning human ventricular cardiomyoeytes with brief periods of stimulated isehemia. Cardiovase Res 1994; 28: 1285～1291
9 Yellon DM, Alkulaifi AM, Pugsley WB. Preconditioning the human myocardium. Lancet 1993; 342: 276～277
10 Yellon DM, Dana A. The preconditioning phenomenon: a tool for the scientist or a clinical reality? Cire Res 2000; 87:543～550
11 Cohen MV, Walsh RS, Goto M, et al. Hypoxia preconditioning rabbit my
ocardium via adenosine and catecholamine release. J Mol Cell Cardiol 1995; 27: 1527～1534
12 Koning MMG, Gho BCG, van Klaarwater E, et al. Rapid ventricular pacing produces myocardial protection by nonischemic activation of K- ATP+channels. Circulation 1996; 93: 178～186
13 Cumming DVE, Heads RJ, Brand NT, et al. The ability of heat stress and metabolic preconditioning to protect primary rat cardiac myocytes. Basic Res Cardiol 1996; 91:79～85
14 Cohen MV, Baines CP, Downey JM. Ischemic preconditioning: from adenosine receptor to KATP channel. Annu Rev Physiol 2000; 62:79～109
15 Cave AC. Preconditioning induced protection against postischemic contractile dysfunction: characteristics and mechanism. J Mol Cell Cardiol 1995; 27: 969～979
16 Nakamura M, Wang N -P, Zhao Z -Q, et al. Preconditioning decreases Bax expression. PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res 2000; 45:661～670
17 Ytrehus K, Liu Y, Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol 1994; 266: H1145～H1152
18 Mortimer SL, Kodl CT, Reiling CR, et al. Preconditioning induced attenuation of purine metabolic accumulation during ischemia: memory and multiple cycles. Basic Res Cardid 2000; 95: 119～126
19 Pan H-L, Chen S-R, Scicli GM, et al. Cardiac interstitial bradykinin release during ischemia is enhanced by ischemic preconditioning. Am J Physiol Heart Circ Physiol 2000; 279:H116～H121
20 Tanno M, Tsuchida A, Nozawa Y, et al. Roles of tyrosine kinase and protein kinase C in infarct size limitation by repetitive isehemic preconditioning in the rat. J Cardiovase Pharmaeol 2000; 35: 345～352
21 Schulz R, Gres P, Heusch G. Role of endogenous opioids in isehemic preconditioning but not in short - term hibernation in pigs. Am J Physiol Heart Cire Physiol 2001; 280:H2175～H2181
22 Pagliaro P, Gattullo D, Rastaldo R, et al. Ischemic preconditioning: from the first to the second window of protection. Life Sci 2001; 69(1): 1～15
23 Vondriska TM, Klein JB, Ping P. Use of functional proteomies to investigate PKCε-mediated cardioprotection: the signaling module hypothesis. Am J Physiol Heart Circ Physiol 2001; 280: H1434～H1441
24 Tani M, Honma Y, Hasegawa H, et al. Direct activation of mitochondrial K(ATP) channels mimic preconditioning but protein kinase C activation is less effective in middle - aged rat hearts. Cardiovasc Res 2001; 49 (1): 56～68
25 Nakano A, Baines CP, Kim So, et al. Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart. Evidence for involvement of p38MAPK. Circ Res 2000; 86: 144～154
26 Chevalier D, Allen BG. Two distinct forms of MAPKAP kinase -2 in adult cardiac ventricular myocytes. Biochemistry 2001; 39: 6145～6156
27 Lonchner A, Genade S,Tromp E, et al. Ischemic preconditioning and the β- adrenergic signal transduetion pathway. Circulation 1999;100:958～966
28 张书岭，尹桂山．组织匀浆腺苷酸环化酶的简易测定法．河北医学院学报1991；12(2)：18～20
29 苗明三主编．实验动物和动物实验技术．中国中医药出版社1997年6月第一版，193～194
30 陈红，章同华，苏定冯．大鼠急性心肌梗塞及缺血／再灌注损伤模型．第二军医大学学报1992；13(2)：176～178
31 Horneffer PJ, Healy B, Gott VL, et al. The rapid evaluation of a myocardial infarction in an end- artery coronary preparation. Circulation 1987; 76 (supple 5): V39～V42
32 Fishbein MC, Meerbaum S, Rit J, et al. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J 1981; 101: H593～H600
33 Vivaldi MT, Kloner RA, Sehoen FJ. Triphenyltetrazolium staining of irreversible ischemie injury following coronary artery occlusion in rats. Am J
Pathol 1985; 121: 522～530
34 Parikh V, Singh M. Possible role of cardiac mast cell degraulation and preservation of nitric oxide release in isolated rat heart subjected to ischemic preconditioning. Mol Cell Biochem 1999; 199:1～6
35 Mosca SM, Gelpi RI, Cingolani HE. Adenosine and dipyridamole mimic the effects of ischemie preconditioning. J Mol Cell Cardiol 1994; 26:1403～1409
36 Kobara M, Tatsumi T, Matoba S, et al. Effect of isehemie preconditioning on mitoehondrial oxidative phosphorylation and high energy phosphates in rat hearts. J Mol Cell Cardiol 1996; 28:417～428
37 兰晓莉，裴著果．受体在心肌缺血预适应中的作用．心脏杂志2001；13(6)：592～595
38 Maisel AS, Ransnas LA, Insel PA. Beta -adrenergie receptor and the Gs protein in myocardial ischemie and injury. Basic Res Cardiol 1990; 85(supple 1): 47～56
39 Maisel AS, Motulsky HJ, Insel PA. Externalization of beta - adrenergie receptors promoted by myocardial ischemia. Science 1985; 230(4722): 183～186
40 Yabe KI, Ishishita H,Tanonaka K, et al. Pharmacologic preconditioning induced by β-adrenergic stimulation is mediated by activation of protein kinase C. J Cardiovasc Pharmaeol 1998 ;32:962～968
41 Wolfe CL,Sievers RE,Visseren FLJ,et al. Loss of myocardial protection after preconditioning correlates with the time course of glycogen recovery within the preconditioned segment. Circulation 1993;87:881～892
42 Strasser RH, Marquetact R. Supersensitivity of the adenylyl cyclase system in acute myocardial isehemia: evaluation of three independent mechanism. Basic Res Cardiol 1990; 85 (supple 1): 67～78
43 Moolman JA, C, enade S, Tromp E, et al. A comparison between ischemic preconditioning and anti - aclrenem'gic interventions: cAMP, energy metabolism and functional recovery. Basic Res Cardiol 1996; 78:137～147
44 Tomoyuki I,Tomoyuki M,Tetsuya T, et al. Ischemic preconditioning is as
sociated with a delay in ischemic - induced reduction of β-adrenergic signal transduction in rabbit hearts. Circulation 1993;88:2827～2837
45 Mieno S, Horimoto H, Watanabe F, et al. Potent adenylate cyelase agonist forskolin restores myoprotective effects of ischemic preconditioning in rat hearts after myocardial infarction. Ann Thorac Surg 2002; 74: 1213～1218
46 Okruhbicova L, Ravingerova T, Pancza D, et al. Activation of adenylate cyclase system in the preconditioning rat heart. Physiol Res 2000; 49: 251～259
47 Sandhu R,Thomas U, Diaz RJ, et al. Effect of ischemic preconditioning of the myocardium on cAMP. Cite Res 1996;78:137～147
48 Lonchner A, Genade S,Tromp E,et al. Role of cyclic nucleotide phosphodiesterases in ischemic preconditioning. Mol Cell Biochem 1998;186:169～175
49 Netticadan T,Temsab R, Osada M, et al. Status of Ca~(2+)/Calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia -reperfusion. Am J Physiol 1999;277(3pt2):C 384～391
50 Osada M, Netticadan J, Tamura K, et al. Modification of ischemic-reperfusion -induced changes in cardiac sarcoplasmic reticulum by preconditioning. Am J Physiol 1998; 274(43): H2025～H2034
51 Persad S, Takeda S, Panagia V, et al. β-adrenoceptor- linked signal transduction in ischemic - reperfusion heart and scavenging of oxyradicals. J Mol Cell Cardiol 1997; 29:545～558
52 Sasaki T, Inui M, Kimura Y, et al. Molecular mechanism of regulation of Ca~(2+) pump ATPase by phopholamban in cardiac sareoplasmic reticulum. Effects of synthetic phospholamban peptides on Ca~(2+) pump ATPase. J Biol Chem 1992; 267: 1674～1679
53 Kargacin ME, Ali Z, Kargaein G. Anti -phospholaban and protein kinase A alter the Ca~(2+) sensitivity and maximum relocity of Ca~(2+) uptake by the cardiac sareoplasmic reticulum. Bioehem J 1998; 331: 245～249
54 Xu M, Wang Y, Hirai K, et al. Calcium preconditioning inhibits rnitochondrail permeability transition and apoptosis. Am J Physiol Heart Circ Physiol
2001;280(2): H899～908
55 Sanda S, Kitakaze M, Papst PJ, et al. Cardioprotection effect afforded by transient exposure to phosphodiesterase Ⅲ inhibitors. The role of protein kinase A and p38 Mitogen- Activated protein kinase. Circulation 2001; 104:705～710
56 Nakano A, Baines CP, Kim CO, et al. Ishemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38MAPK. Circ Res 2000; 86:144～151
57 Sanda S, Kitakaze M, Papst PJ, et al. Role of phasic dynamism of p38 Mitogen - Activated protein kinase activation in ischemic preconditioning of the canine heart. Circ Res 2001;88:175～180
58 Bluhm WF, Martin JL, Mestril R, et al. Specific heat shock proteins protect mierotubules during simulated ischemia in cardiac myoeytes. Am J Physiol 1998; 275: H2243～H2249
59 Baines CP, Liu GS, Birineioglu M, et al. Ischemic preconditioning depends on interaction between mitochodrial KATP channels and actin cytoskeleton. Am J Physiol 1999; 276: H1361～H1368
2 Kaszala K, Vegh A, Papp JG, et al. Time course of the protection against ischemia and repeffusion - induced ventricular arrhythmias resulting from brief period of cardiac pacing. J Mol Cell Cardiol 1996; 28:2085～2095
3 Mullane K, Bullough D. Harnessing an endogenous cardioprotective mechanism: sources and sites of action of adenosine. J Mol Cell Cardiol 1995; 27: 1041～1054
4 Wang G - Y, Wu S, Pei J - M, et al. K but not δ - opioid receptors mediate effects of isehemic preconditioning on both infarct and arrhythmia in rats. Am J Physiol Heart Circ Physiol 2001; 280: H384～H391
5 Lan X - L, Diao Y, Lan J - Ch, et al. The effect of aclrenergie receptor-adenyl cyclase system on myocardial ischemie preconditioning in rats. Nuclear Science and Techniques 2002; 13(4): 230～236
6 Schott RJ, Rohmann S, Braun ER, et al. Isehemie preconditioning reduces infarct size in swine myocardium. Circ Res 1990; 66:1133～1142
7 Morris SD, Yellon DM. Angiotension -converting enzyme inhibitors potentiate preconditioning through bradykinin B2 receptor activation in human heart. JACC 1997; 29:1599～1606
8 Ikonomidis JS, Tumiati LC, Weisel RD, et al. Preconditioning human ventricular cardiomyoeytes with brief periods of stimulated isehemia. Cardiovase Res 1994; 28: 1285～1291
9 Yellon DM, Alkulaifi AM, Pugsley WB. Preconditioning the human myocardium. Lancet 1993; 342: 276～277
10 Yellon DM, Dana A. The preconditioning phenomenon: a tool for the scientist or a clinical reality? Cire Res 2000; 87:543～550
11 Cohen MV, Walsh RS, Goto M, et al. Hypoxia preconditioning rabbit my
ocardium via adenosine and catecholamine release. J Mol Cell Cardiol 1995; 27: 1527～1534
12 Koning MMG, Gho BCG, van Klaarwater E, et al. Rapid ventricular pacing produces myocardial protection by nonischemic activation of K- ATP+channels. Circulation 1996; 93: 178～186
13 Cumming DVE, Heads RJ, Brand NT, et al. The ability of heat stress and metabolic preconditioning to protect primary rat cardiac myocytes. Basic Res Cardiol 1996; 91:79～85
14 Cohen MV, Baines CP, Downey JM. Ischemic preconditioning: from adenosine receptor to KATP channel. Annu Rev Physiol 2000; 62:79～109
15 Cave AC. Preconditioning induced protection against postischemic contractile dysfunction: characteristics and mechanism. J Mol Cell Cardiol 1995; 27: 969～979
16 Nakamura M, Wang N -P, Zhao Z -Q, et al. Preconditioning decreases Bax expression. PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res 2000; 45:661～670
17 Ytrehus K, Liu Y, Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol 1994; 266: H1145～H1152
18 Mortimer SL, Kodl CT, Reiling CR, et al. Preconditioning induced attenuation of purine metabolic accumulation during ischemia: memory and multiple cycles. Basic Res Cardid 2000; 95: 119～126
19 Pan H-L, Chen S-R, Scicli GM, et al. Cardiac interstitial bradykinin release during ischemia is enhanced by ischemic preconditioning. Am J Physiol Heart Circ Physiol 2000; 279:H116～H121
20 Tanno M, Tsuchida A, Nozawa Y, et al. Roles of tyrosine kinase and protein kinase C in infarct size limitation by repetitive isehemic preconditioning in the rat. J Cardiovase Pharmaeol 2000; 35: 345～352
21 Schulz R, Gres P, Heusch G. Role of endogenous opioids in isehemic preconditioning but not in short - term hibernation in pigs. Am J Physiol Heart Cire Physiol 2001; 280:H2175～H2181
22 Pagliaro P, Gattullo D, Rastaldo R, et al. Ischemic preconditioning: from the first to the second window of protection. Life Sci 2001; 69(1): 1～15
23 Vondriska TM, Klein JB, Ping P. Use of functional proteomies to investigate PKCε-mediated cardioprotection: the signaling module hypothesis. Am J Physiol Heart Circ Physiol 2001; 280: H1434～H1441
24 Tani M, Honma Y, Hasegawa H, et al. Direct activation of mitochondrial K(ATP) channels mimic preconditioning but protein kinase C activation is less effective in middle - aged rat hearts. Cardiovasc Res 2001; 49 (1): 56～68
25 Nakano A, Baines CP, Kim So, et al. Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart. Evidence for involvement of p38MAPK. Circ Res 2000; 86: 144～154
26 Chevalier D, Allen BG. Two distinct forms of MAPKAP kinase -2 in adult cardiac ventricular myocytes. Biochemistry 2001; 39: 6145～6156
27 Lonchner A, Genade S,Tromp E, et al. Ischemic preconditioning and the β- adrenergic signal transduetion pathway. Circulation 1999;100:958～966
28 张书岭，尹桂山．组织匀浆腺苷酸环化酶的简易测定法．河北医学院学报1991；12(2)：18～20
29 苗明三主编．实验动物和动物实验技术．中国中医药出版社1997年6月第一版，193～194
30 陈红，章同华，苏定冯．大鼠急性心肌梗塞及缺血／再灌注损伤模型．第二军医大学学报1992；13(2)：176～178
31 Horneffer PJ, Healy B, Gott VL, et al. The rapid evaluation of a myocardial infarction in an end- artery coronary preparation. Circulation 1987; 76 (supple 5): V39～V42
32 Fishbein MC, Meerbaum S, Rit J, et al. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J 1981; 101: H593～H600
33 Vivaldi MT, Kloner RA, Sehoen FJ. Triphenyltetrazolium staining of irreversible ischemie injury following coronary artery occlusion in rats. Am J
Pathol 1985; 121: 522～530
34 Parikh V, Singh M. Possible role of cardiac mast cell degraulation and preservation of nitric oxide release in isolated rat heart subjected to ischemic preconditioning. Mol Cell Biochem 1999; 199:1～6
35 Mosca SM, Gelpi RI, Cingolani HE. Adenosine and dipyridamole mimic the effects of ischemie preconditioning. J Mol Cell Cardiol 1994; 26:1403～1409
36 Kobara M, Tatsumi T, Matoba S, et al. Effect of isehemie preconditioning on mitoehondrial oxidative phosphorylation and high energy phosphates in rat hearts. J Mol Cell Cardiol 1996; 28:417～428
37 兰晓莉，裴著果．受体在心肌缺血预适应中的作用．心脏杂志2001；13(6)：592～595
38 Maisel AS, Ransnas LA, Insel PA. Beta -adrenergie receptor and the Gs protein in myocardial ischemie and injury. Basic Res Cardiol 1990; 85(supple 1): 47～56
39 Maisel AS, Motulsky HJ, Insel PA. Externalization of beta - adrenergie receptors promoted by myocardial ischemia. Science 1985; 230(4722): 183～186
40 Yabe KI, Ishishita H,Tanonaka K, et al. Pharmacologic preconditioning induced by β-adrenergic stimulation is mediated by activation of protein kinase C. J Cardiovasc Pharmaeol 1998 ;32:962～968
41 Wolfe CL,Sievers RE,Visseren FLJ,et al. Loss of myocardial protection after preconditioning correlates with the time course of glycogen recovery within the preconditioned segment. Circulation 1993;87:881～892
42 Strasser RH, Marquetact R. Supersensitivity of the adenylyl cyclase system in acute myocardial isehemia: evaluation of three independent mechanism. Basic Res Cardiol 1990; 85 (supple 1): 67～78
43 Moolman JA, C, enade S, Tromp E, et al. A comparison between ischemic preconditioning and anti - aclrenem'gic interventions: cAMP, energy metabolism and functional recovery. Basic Res Cardiol 1996; 78:137～147
44 Tomoyuki I,Tomoyuki M,Tetsuya T, et al. Ischemic preconditioning is as
sociated with a delay in ischemic - induced reduction of β-adrenergic signal transduction in rabbit hearts. Circulation 1993;88:2827～2837
45 Mieno S, Horimoto H, Watanabe F, et al. Potent adenylate cyelase agonist forskolin restores myoprotective effects of ischemic preconditioning in rat hearts after myocardial infarction. Ann Thorac Surg 2002; 74: 1213～1218
46 Okruhbicova L, Ravingerova T, Pancza D, et al. Activation of adenylate cyclase system in the preconditioning rat heart. Physiol Res 2000; 49: 251～259
47 Sandhu R,Thomas U, Diaz RJ, et al. Effect of ischemic preconditioning of the myocardium on cAMP. Cite Res 1996;78:137～147
48 Lonchner A, Genade S,Tromp E,et al. Role of cyclic nucleotide phosphodiesterases in ischemic preconditioning. Mol Cell Biochem 1998;186:169～175
49 Netticadan T,Temsab R, Osada M, et al. Status of Ca~(2+)/Calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia -reperfusion. Am J Physiol 1999;277(3pt2):C 384～391
50 Osada M, Netticadan J, Tamura K, et al. Modification of ischemic-reperfusion -induced changes in cardiac sarcoplasmic reticulum by preconditioning. Am J Physiol 1998; 274(43): H2025～H2034
51 Persad S, Takeda S, Panagia V, et al. β-adrenoceptor- linked signal transduction in ischemic - reperfusion heart and scavenging of oxyradicals. J Mol Cell Cardiol 1997; 29:545～558
52 Sasaki T, Inui M, Kimura Y, et al. Molecular mechanism of regulation of Ca~(2+) pump ATPase by phopholamban in cardiac sareoplasmic reticulum. Effects of synthetic phospholamban peptides on Ca~(2+) pump ATPase. J Biol Chem 1992; 267: 1674～1679
53 Kargacin ME, Ali Z, Kargaein G. Anti -phospholaban and protein kinase A alter the Ca~(2+) sensitivity and maximum relocity of Ca~(2+) uptake by the cardiac sareoplasmic reticulum. Bioehem J 1998; 331: 245～249
54 Xu M, Wang Y, Hirai K, et al. Calcium preconditioning inhibits rnitochondrail permeability transition and apoptosis. Am J Physiol Heart Circ Physiol
2001;280(2): H899～908
55 Sanda S, Kitakaze M, Papst PJ, et al. Cardioprotection effect afforded by transient exposure to phosphodiesterase Ⅲ inhibitors. The role of protein kinase A and p38 Mitogen- Activated protein kinase. Circulation 2001; 104:705～710
56 Nakano A, Baines CP, Kim CO, et al. Ishemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38MAPK. Circ Res 2000; 86:144～151
57 Sanda S, Kitakaze M, Papst PJ, et al. Role of phasic dynamism of p38 Mitogen - Activated protein kinase activation in ischemic preconditioning of the canine heart. Circ Res 2001;88:175～180
58 Bluhm WF, Martin JL, Mestril R, et al. Specific heat shock proteins protect mierotubules during simulated ischemia in cardiac myoeytes. Am J Physiol 1998; 275: H2243～H2249
59 Baines CP, Liu GS, Birineioglu M, et al. Ischemic preconditioning depends on interaction between mitochodrial KATP channels and actin cytoskeleton. Am J Physiol 1999; 276: H1361～H1368