微小RNA在心肌缺血再灌注中的临床和基础研究
|
摘要
研究背景：
冠心病是导致人类死亡的主要病因之一,因为心肌缺血损伤和缺血再灌注损伤导致患者死亡或心力衰竭发生率非常高。对于急性心肌梗死患者,利用溶栓或早期用经皮冠状动脉介入治疗进行有效的心肌再灌注是缩小心肌梗死面积,改善临床转归的有效方法。但是对于心肌缺血后损伤和缺血再灌注损伤对心功能的影响和突发心脏事件的发生如何预测和减低是非常棘手的问题。尽管我们对心肌缺血再灌注损伤的机制目前研究发现主要认为是中性粒细胞聚集、钙超载或钙再分布、线粒体能量合成障碍、氧自由基生成、细胞因子、心肌细胞凋亡等现象所引起的,但是我们是否能对引起这些变化的相关调控物质予以检测和干预来达到理想的效果呢?
缺血是由于供血障碍引起的组织供血不足的病理表现,组织缺血导致的疾病称缺血性疾病,是可以发生在多种组织器官上的常见疾病。再灌注是指组织缺血后恢复供血的过程,缺血再灌注实际上包括缺血和再灌注两个方面,缺血是引起疾病的原因,,也是再灌注的条件。及时的心肌再灌注治疗,如溶栓、经皮冠状动脉介入治疗(PCD等都可以重建冠状动脉血运,使缺血心肌得到再灌注,从而减少心肌坏死面积,是急性心肌梗死(AMI)治疗的关键。但近来研究发现,冠状动脉的骤然开通与血流恢复,会引起血管内皮细胞功能异常、激活相关炎症因子等,反而会加重缺血心肌的损伤,导致再灌注损伤。AMI的动物研究结果已经表明,40%-50%的坏死心肌为再灌注损伤所引起,这种损伤的存在减弱了积极再灌注治疗所得到的益处。因此,减少再灌注损伤成为AMI防治的非常重要环节之一。
心肌缺血后损伤和缺血再灌注损伤目前主要有以下几种观点：1、自由基损伤学说。当机体损伤产生的自由基超过机体的清除能力时,将对机体产生损伤作用。研究证实缺血再灌注过程中,缺血区有大量的自由基产生,如清除氧自由基能力降低或活性氧自由基(ROI)产生过多,都会导致ROI蓄积,从而引发心肌氧化应激。氧化应激是缺血组织再灌注的特征之一。而且研究证实应用自由基清除剂辅酶Q10可以减轻缺血再灌区细胞的损伤。2、钙超载是造成心肌细胞损伤的重要机制之一,缺血缺氧时心肌细胞内酸中毒,细胞内H+增加,恢复再灌注时细胞内外形成pH梯度差,刺激Na+-H+交换,使Na+大量内流。再灌注后由于能量供应和pH值的恢复,促进Na+-Ca2+交换,,细胞外的Ca2+大量内流,,造成Ca2+超载。从而使细胞膜和细胞器膜受损、促进氧自由基生成而导致心肌损伤。同时由于缺血时血管平滑肌有明显Ca2+内流增加,可致血管收缩痉挛,血管阻力增加对缺血区循环不利,使心肌梗死灶扩大。研究发现,细胞内钙离子浓度的改变是造成再灌注损伤的重要原因之一,钙超载还是多种原因导致的细胞损伤和死亡的共同通路。3、心肌能量代谢障碍。三磷酸腺苷(ATP)是细胞代谢的主要能量来源,细胞所处的生命状态和一切生命活动最终都依赖AT P的水平。如果轻度缺氧或短暂严重缺氧后复氧,细胞仍可生成部分ATP,可为凋亡所需能量提供保障,促使细胞发生凋亡。反之,如果较长时间严重缺氧,细胞由于AT P骤减导致凋亡所需能量不足而发生坏死；当ATP生成被完全抑制后细胞发生坏死,提示ATP在缺氧诱导细胞死亡过程中是细胞死亡方式的决定性因素之一。能量代谢障碍也是自由基产生的基础,自由基损伤又可加重能量代谢障碍,两者也是互为因果的关系。所以说,线粒体损伤和能量代谢障碍是心肌缺血再灌注损伤的重要原因,细胞内的AT P水平是决定细胞发生凋亡或坏死的主要因素。4、内皮细胞抗氧化系统损伤,研究证明内皮细胞的抗氧化活性与其对氧化活性的敏感性之间有一定的关系。心肌缺血再灌注时,内皮细胞不仅产生大量的活性氧,而且其抗氧化活性大大降低,并对外源性的活性氧产生系统有较高的敏感性,从而引起大量活性氧的产生,远远超过了内皮细胞的防御系统,最终引起心肌的损伤。有研究表明,缺血再灌注发生的内皮细胞功能障碍及NO合成的减少,内皮素(ET)心肌缺血时ET释放增加,再灌注时ET释放进一步增加,引起了强烈的血管收缩,可直接引起心肌缺血,加重血管功能障碍,使细胞发生不可逆的损伤。组织缺氧及肾上腺素分泌增加均可导致ET的mRNA表达及ET释放,心肌缺血再灌注还可引起心肌细胞膜ET受体上调,并使冠状血管对ET的敏感性增加,冠脉小分支因而易于痉挛,可导致心肌的无复流现象。内皮细胞与中性粒细胞粘附缺血、再灌注引起中性粒细胞与内皮细胞的粘附增加,中性粒细胞与血管内皮接触时即被激活,释放OFR等毒性产物及破坏性蛋白酶,改变血管的通透性。同时活性氧产生的增加,损伤了内皮细胞和其他细胞,其作用于内皮细胞或粒细胞表面的粘附分子,促进内皮细胞的粘附,而缺氧本身也损伤内皮功能,改变其粘附性,最终导致内皮细胞破损、水肿和功能障碍,毛细血管腔被阻塞,导致虽有大血管的再灌注但局部缺血区仍无复流的现象。5、相关细胞因子作用。由于缺氧复氧的刺激,冠状血管受损,这时肿胀的内皮细胞阻碍了气体交换,而内皮细胞和平滑肌细胞的损伤使血管不能很好扩张,血管腔内中性粒细胞和血小板的聚集通过嵌顿、堵塞毛细血管,使冠脉血流更趋减少；继而血管内皮和中性粒细胞产生细胞因子、粘附因子,使中性粒细胞向血管内皮细胞粘附,并释放颗粒状弹性硬蛋白、活性氧、溶酶体酶、细胞因子和其他炎性介质。这些物质会损伤内皮、血管平滑肌细胞和心肌细胞。6、细胞凋亡参与了这一过程,细胞凋亡是缺血再灌注组织损伤功能丧失的重要原因,它是一个很复杂的过程。细胞凋亡的诱导因素很多,而在心肌缺血再灌注引起的细胞凋亡中主要机制则为自由基增多和细胞内Ca2+水平升高。由于缺血缺氧可使内源性抗氧化剂如SOD失活或耗尽,从而使AT P代谢产物在缺血缺氧心肌组织中堆积,产生大量氧自由基。氧自由基性质极不稳定,一旦形成迅速使肌膜的葡萄糖与脂质过氧化,蛋白质变性,酶失活,并使细胞内的DNA链断裂,从而诱导细胞凋亡。
目前对于微小RNA在心血管系统的作用研究处于热点中。微小RNA(microRNA, miRNA)是近年发现的小的、内源性的单链的非编码RNA,大约由22个核苷酸组成,与基因表达的控制有关。它被认为是基因表达的负面调节器,通过与其目标mRNA分子的3’端非编码区互补匹配导致该mRNA分子的翻译受到抑制。目前大约有三分之一的基因受miRNA调控。最近的研究表明一些miRNA在心血管高度表达,它们在调控心血管的发育和疾病方面具有重要意义,这给心血管疾病的治疗带来了希望。研究发现大鼠离体和活体的心肌缺血/再灌注模型中miR-320的表达式持续性失调。鉴定热休克蛋白20(一种已知的心肌保护蛋白)是miR-320的靶体。敲除内源性miR-320发现通过热休克蛋白20来保护有心肌缺血再灌注诱导的心肌细胞死亡/凋亡将失去。另外的研究发现大鼠缺血6小时后心脏的缺血区miR-21的表达是明显下降的,而在周围边缘区表达是上升的。在离体转染miR-21通过其靶基因执行激活蛋白1通路来降低边缘区和缺血区细胞的凋亡。在活体中实验表明miR-1和miR-133对细胞的凋亡是起相反作用的。miR-1还通过抑制胰岛素样生长因子1的转运与细胞的死亡是联系在一起的。最近的研究发现在病人的外周循环血中也可检测出miRNAS。因此,我们可以假设外周血中miRNAs反映了组织的损害,基于此miRNAs可以作为急性心肌梗死的血清生物标记物。
miR-126(microRNA-126)作为miRNAs家族中的一员,人们通过对小鼠动脉粥样硬化模型的研究,发现其在其中有调节细胞凋亡的作用。在人的内皮细胞中,对血管的形成起副调节作用,抑制细胞衍生因子1的表达。调节血管的完整性、血管内皮细胞的增值和新生血管的形成。Sun X等研究认为miR-126的下调和上调和冠心病发病无明显相关,只是和胆固醇的代谢有相关性,在高的冠心病伴低密度胆固醇(LDL)的患者中的表达量是减少的。但是Guangwen Long等在急性心肌梗死中测得血浆中的miR-126表达和急性心肌梗死有关,在急性心肌梗死中的miR-126表达是减少的,且其上调和下调与时间相关,其在不同时间的表达曲线基本是和血清肌钙蛋白Ⅰ的时间表达曲线是一致的,但彼此未见明显相关性。miR-92a作为miRNAs家族中的另一员,研究发现在人的内皮细胞中高度表达,控制新的血管生成。体内和体外的研究发现miR-92a在内皮细胞中的过度表达损害血管生成。离体的研究表明诱导缺血后组织中的miR-92a的表达明显上调。在急性心肌梗死模型中抑制miR-92a的表达回促进心脏功能的恢复。实验研究表明miR-92a不仅在内皮细胞中表达,而且在心脏成纤维细胞、心肌细胞中,拮抗niR-92a能明显降低细胞凋亡,但并没有影响细胞的生存。研究还发现miR-92a依赖性调节NO合酶来控制血管张力。研究表明在冠心病患者中的表达偏高,他汀类可以通过减低miR-92a表达来减低血脂。抑制了miR-92a可能会减少心肌梗死面积,改善心肌梗死后重塑和新生血管。在心肌缺血／再灌注中研究表明miR-92a通过促进心脏保护蛋白的合成,起到积极作用。因此,本研究从临床和基础方面对miR-92a和miR-126在心肌缺血再灌注中作用进行研究,为未来冠心病的诊治提供方法和措施。
研究目的：
1、通过对急性心肌梗死患者外周血中的miR-92a和miR-126表达研究,检测miR-92a和miR-126能否进一步早期诊断急性心肌梗死；
2、对急性心肌梗死患者行再灌注治疗前后外周血中miR-92a和miR-126表达来阐述微小RNA在其中的可能作用。
3、通过对急性心肌梗死行再灌注治疗后出现无复流现象时miR-92a和miR-126表达的研究,试图说明miR-92a和miR-126在其中的可能作用。
3、通过建立小鼠的心肌缺血再灌注实验研究来推断miR-92a和miR-126在心肌缺血再灌注损伤中可能的调控机制。
研究方法：
1、临床外周血中微小RNA等相关标本的收集和检测
1.1选择了36例证实为冠心病患者和与之年龄向匹配的3例健康者进入临床研究。分组：稳定性心绞痛组、不稳定性心绞痛组、非ST段抬高性急性心肌梗死组、ST段抬高性急性心肌梗死组、健康对照组。所有冠心病患者均行PCI治疗,AMI患者行急诊PCI治疗。
1.2取冠心病患者PCI术前和PCI术后1、5天的静脉血。健康对照组为匹配的时间。将采集好的全血转移至单独的冻存管,短期保存,放入-70℃冰箱,长期保存,放入液氮中。
1.3采用实时定量荧光PCR (qRT-PCR)来测定miR-92a、miR-126的表达,所有检测过程按照上海康成公司提供的试剂盒说明来进行。同时检测患者血中的相关指标：清肌钙蛋白I (cTNI)、N-端脑钠肽前体(NT-proBNP)、超敏C反应蛋白(hs-CRP)、D-D二聚体(D-D)等指标。并分析PCI手术时患者是否有无复流现象。
1.4统计学分析：采用SPSS18.0统计软件。使用t检验或方差分析,参数以x±S表示。相关分析采用Pearson或Spearman相关分析法,P<0.05为差异有统计学意义
2、动物实验中外周血中微小RNA等相关标本的收集和检测
2.1小鼠心肌缺血再灌注模型的建立及分组：
选昆明小鼠结扎其左冠状动脉前降支,以小鼠心肌颜色变白且心电图ST段持续弓背抬高为模型成功标志。结扎30分钟后,撤除结扎线,以心肌恢复红润且ST段下降作为再灌注模型成功标志。目前此动物模型建立是成熟的。分组：假手术组(9只)、模型组(9只)、干预模型组(9只),每组中的3只用制成组织学切片。干预模型组用阿托伐他汀(2mg/kg)每天一次灌胃,其他两组给予等量的生理盐水,共7天。
2.2标本采集和相关指标的完成
各组小鼠分别设计为结扎冠状动脉30分钟后再灌注12h各6只,取部分血用来检测血清中相关指标,另取血400ul,将采集好的全血转移至单独的冻存管,短期保存,放入-70℃冰箱,长期保存,放入液氮中。杀死小鼠后取不同标本(缺血区、边缘区)组织经固定-脱水-石蜡包埋-切片。
2.3检测相关指标
用伊文氏蓝来进行染色。细胞凋亡指数用TUNEL法。全血采用实时定量荧光PCR (qRT-PCR)来测定miR-92a、miR-126的表达。CK-MB用免疫发光法,用化学法测试超氧化物歧化酶(SOD)、丙二醛(MDA)。
2.4统计学分析
采用SPSS13.0统计软件。使用t检验或方差分析,参数以x±S表示。相关分析采用Pearson或Spearman相关分析法,P<0.05为差异有统计学意义
结果：
临床试验的结果
1、qRT-PCR显示,冠心病患者的]miR-92a表达量与健康对照组是增加的,两者之间有显著性差异(P<0.05)；不同类型冠心病患者miR-92a表达量随病情程度加重而升高(P<0.05); PCI术后miR-92a表达量是降低的,PCI术前后miR-92a表达量有显著性差异(P<0.05)；同一类型冠心病患者PCI术后随时间的变化miR-92a表达量降低(P<0.05)。
2、急性心肌梗死患者(包括非ST段抬高性心肌梗死和ST段抬高性急性心肌梗死)的miR-126表达量与健康对照组是降低的,两者之间有显著性差异(P<0.05)；其他类型冠心病患者miR-126表达量与健康对照组无显著性差异(P>0.05)；急性心肌梗死PCI术后miR-126表达量是增加的,PCI术前后miR-126表达量有显著性差异(P<0.05)；同一类型冠心病患者PCI术后随时间的变化miR-126表达量增加(P<0.05)。
3、急性心肌梗死患者血中的血清肌钙蛋白I (cTNI)、超敏C反应蛋白(hs-CRP)、D-D二聚体(D-D)的水平,发现这些指标均与miR-92a表达量无统计学意义的相关关系(r=0.162, P=0.515; r=0.424, P=0.079; r=0.361, P=0.141)。N-端脑钠肽前体(NT-proBNP)与miR-92a表达量有统计学意义的相关关系(r=0.583,P=0.011)。N-端脑钠肽前体(NT-proBNP)、超敏C反应蛋白(hs-CRP)、D-D二聚体(D-D)的水平均与miR-126表达量有统计学意义的相关关系(r=-0.605, P=0.033; r=-0.666, P=0.008; r=-0.616, P=0.008)。血清肌钙蛋白I (cTNI)与miR-126表达量无统计学意义的相关关系(r=-0.368,P=0.133)。
4、急性心肌梗死患者再灌注治疗出现无复流现象分析：(1)miR-126表达量、miR-92a表达量、hs-CRP水平、D-D水平在两组之间有显著性差异(P<0.05)。(2)miR-126表达量与hs-CRP水平、D-D水平有统计学意义的相关关系(r1=-0.666,r2=-0.616,P<0.05),miR-92a表达量与hs-CRP水平、D-D水平有统计学意义的相关关系(r1=0.638,r2=0.623,P<0.05)。
动物实验的结果
1、通过使用qRT-PCR法检测出的小鼠心肌缺血再灌注不同组别(假手术组、缺血再灌注组、药物干预再灌注组)中全血中的：miR-92a.miR-126相对表达量(2-△△CT),经过分析：两者在假手术组、缺血再灌注组、药物干预组的表达量有显著性差异(P<0.05)
2、假手术组、缺血再灌注组、药物干预再灌注组三组中miR-92a和miR-126和血清标志物及心肌细胞凋亡指数之间的相关关系：(1)miR-92a和CK-MB、MDA的变化是一致,而与SOD的变化是相向的。(2)miR-126表达量的变化和CK-MB、MDA的变化是相反,而与SOD的变化是一致的。(3)miR-92a表达量变化心肌细胞凋亡指数的变化趋势是一致的。而miR-126表达量的变化和心肌细胞凋亡指数的变化趋势是相反的。
结论：
1、发现miR-92a和miR-126参与了心肌缺血再灌注的过程。可能为早期诊断和预测无复流现象及可能提前干预提供思路。
2、在心肌缺血再灌注损伤中,发现miR-92a和miR-126的表达与氧自由基和心肌细胞凋亡有关联。
3、他汀类药物的干预可以改变miR-92a和miR-126的表达。
Background
Coronary heart disease is one of the major reasons of human death, because myocardial ischemic injury and ischemia-reperfusion Injury can lead to death or very high incidence of heart failure. For patients with acute myocardial infarction, the using of thrombolysis or percutaneous coronary intervention in the early treatment can reduce myocardial infarct size after the myocardial reperfusion and can be an effective way to improve clinical outcome. However, it is very difficult how to predict and reduce damage after myocardial ischemia and ischemia-reperfusion injury of cardiac function and sudden cardiac events. By studying the mechanism about myocardial ischemia and reperfusion injury, we found that the main reasons are neutrophil accumulation, calcium overload or calcium redistribution, of mitochondrial energy synthesis barriers,the generation of oxygen free radicals. But can we achieve the desired effect by studying these changes with regulators detection and intervention? Recently it is the hotspot research to study the role of microRNA in the cardiovascular system.
Ischemia is a blood disorder caused by insufficient blood supply to tissue pathology. Tissue ischemia caused by the disease known as ischemic disease, it can happen in a variety of tissues and organs of the common diseases.
Reperfusion after ischemia refers to the organization of the process to restore the blood supply.
Ischemia-reperfusion actually includes two aspects.One is ischemia and another is reperfusion. The cause of the disease is not only caused by ischemia, but also the conditions of reperfusion. Timely reperfusion therapy, such as thrombolysis, percutaneous coronary intervention (PCI), etc. can be reconstructed coronary revascularization, myocardial reperfusion obtained. Thereby.reducing the area of myocardial necrosi is the key way of the treatment about acute myocardial infarction (AMI). However, recent studies have found that a sudden opening of the coronary arteries and restore blood flow might cause endothelial cell dysfunction, activation of inflammatory factors. But increasing ischemic myocardial injury could result in reperfusion injury. Animal with AM study results I show that40%to50%of myocardial necrosis is caused by reperfusion injury and this injury weakens the active presence of reperfusion therapy benefits obtained. Therefore, it is to reduce reperfusion injury that become an important part of prevention and treatment of AMI.
Injury after myocardial ischemia and ischemia-reperfusion injury is currently mainly the following points:1, free radical damage theory. When the body produces free radicals damage exceeding the body's ability to clear, the body will produce injury. Study confirmed during ischemia-reperfusion, ischemic area there are plenty of free radicals, such as reduced ability to scavenge oxygen free radicals or reactive oxygen species (ROI). ROI can produce too much will lead to ROI accumulation caused by oxidative stress. Oxidative stress reperfusion of ischemic tissue is one of the characteristics. And the application of free radical scavengers Coenzyme Q10can reduce ischemic cell damage irrigation.
2, Calcium overload caused by myocardial injury is an important mechanism of ischemic hypoxia.The myocardial intracellular acidosis, intracellular H+to increase and restore reperfusion intracellular formation of pH gradient difference, stimulating Na+-H+exchange, so that Na+a large influx. Since the energy supply after reperfusion and pH recovery, promote Na+-Ca2+exchange, a large number of extracellular Ca2+influx, resulting in Ca2+overload. So that the cell membrane and organelle membrane damage caused by oxygen free radicals promote myocardial injury. Ischemic vascular smooth muscle and because a significant increase in Ca2+influx can cause vasoconstriction spasm, vascular resistance increased circulating adverse ischemic area, expand the infarct. Study found that the changes of the concentration of intracellular calcium are caused by reperfusion injury.It is one of the important reasons. Calcium overload is a variety of causes cell damage and death common pathway.
3,Myocardial energy is metabolism, ATP is the main energy source of cell metabolism, cell in which the life of the state and all life activities ultimately depend AT P levels. If mild or transient hypoxia reoxygenation after severe hypoxia, cells can still generate some ATP and the energy required for apoptosis to provide protection to promote apoptosis. Conversely, if a longer time severe hypoxia,cell apoptosis due to AT P plummeted lead to insufficient energy needed necrosis; When ATP production was completely inhibited cell necrosis, Study suggests that hypoxia-induced ATP in the cell during cell death manner of death of the decisive factors. Energy metabolism is the basis of free radicals, free radicals can also increase energy metabolism, they are mutually reinforcing relationship. Therefore, mitochondrial damage and energy metabolism of myocardial ischemia-reperfusion injury is a major cause of intracellular AT P. It is to determine the level of apoptosis or necrosis of the main factors.
4,Anti-oxidation system damage endothelial cells. Studies have shown that the antioxidant activity of endothelial cells and their sensitivity to oxidation activity relationship between certain. When myocardial ischemia and reperfusion, the endothelial cells not only produce large amounts of reactive oxygen species, and also greatly reduce its antioxidant activity.Exogenous reactive oxygen generation system has high sensitivity. It can cause a large number of reactive oxygen species, far endothelial cells than defense system, and ultimately lead to myocardial injury. Other studies have shown that ischemia-reperfusion endothelial cell dysfunction occurs and the reduction of NO synthesis,. Endothelin (ET) ET increase release of myocardial ischemia. Reperfusion ET release further increased causing intense vasoconstriction.It can be directly cause myocardial ischemia, increased vascular dysfunction, cell irreversible damage. Tissue hypoxia and increased secretion of adrenaline can lead to the ET mRNA and ET releasing. Myocardial ischemia and reperfusion can cause myocardial cell ET receptors, and makes the ET coronary vessels increased sensitivity to small branches. And thus coronary Easy spasms that can lead to myocardial no-reflow phenomenon. Endothelial cells and neutrophil adhesion ischemia, reperfusion induced neutrophil adhesion to endothelial cells increased neutrophils and endothelial contact is activated, the release of OFR and other toxic products and destructive proteases, vascular permeability changes. At the same time an increasing in reactive oxygen species will damage to the endothelial cells and other cells, and its role in endothelial cells or granulocyte cell surface adhesion molecules, promote endothelial cell adhesion and endothelial function hypoxic injury itself. Changing its adhesion eventually leading to endothelial cell damage, edema and dysfunction. Capillary chamber is blocked, leading to the great vessels although the area of ischemia-reperfusion, but still no reflow phenomenon.
5, the related cytokines. Bing stimulation of the hypoxia when hindered gas exchange, coronary vascular will injury swelling of endothelial cells. And endothelial cells and smooth muscle cell damage the blood vessels are not well expanding the endovascular neutrophil and platelet aggregation through incarceration, clogging the capillaries, so that more reduced coronary blood flow;Followed by vascular endothelium and neutrophils produce cytokines, adhesion molecules, the neutrophils to vascular endothelial cells stick attached, and the release of particulate elastin, reactive oxygen species, lysosomal enzymes, cytokines and other inflammatory mediators. These substances can damage the endothelium, vascular smooth muscle cells and cardiac myocytes.
6.Involving in the process of cell apoptosis. The apoptosis is ischemia-reperfusion tissue injury in important cause loss of function. It is a very complicated process. Cell apoptosis is Inducted by many factors. And in myocardial ischemia and reperfusion inducing cell apoptosis was the main mechanism.Intracellular free radicals increased Ca2+levels. Because ischemic hypoxia endogenous antioxidants such as SOD inactivation or depletion, AT P metabolites accumulate in hypoxic-ischemic myocardial tissu eand result in a large number of oxygen free radicals. Unstable nature of oxygen free radicals once formed to intracellular DNA strand breaks quickly make sarcolemma glucose and lipid peroxidation, protein denaturation and enzyme inactivation, and can induce apoptosis.
MicroRNAs is a recently discovered small endogenous non-coding single-stranded RNA(about22nucleotides) and the control of gene expression. It is considered to be a negative regulator of gene expression, via its target mRNA molecule3'end complementary to the non-coding region match by inhibiting translation of the mRNA molecule (1-3). Currently, there is about one-third of the genes regulated by MicroRNAs. Recent research suggests that some MicroRNAs are highly expressed in the cardiovascular and they are of great significance in the regulation of cardiovascular development and disease, which will bring hope to the treatment of cardiovascular diseases. The study found that the persistent disorders of miR-320expression in model of the rats away from the body and in vivo myocardial ischemia/reperfusion.They identify that heat shock protein20(a known myocardial protection protein) is the target body of miR-320. After knockdown of endogenous miR-320, the heat shock protein20will lose the protection myocardial ischemia-reperfusion-induced myocardial cell death/apoptosis. Another study found that the miR-21Expression of the ischemic area of the heart is decreased after six hours of ischemia in rats, while the expression is increased in the peripheral edge area. Transfected with miR-21in vitro through the implementation of its target gene activator protein1pathway to reduce cell apoptosis in the edge of the area and the ischemic area. In the vivo experiments of MiR-1and miR-133show that the effect of apoptosis is counterproductive. MiR-1is linked to cell death by inhibiting the transporter of insulin-like growth factor1. Recently study found that miRNAs can be detected in the blood of the patient's peripheral circulation. Therefore, we can assume that the peripheral blood miRNAs reflect damage of tissue. So the detected miRNAs can be serum biomarkers as acute myocardial infarction,.
As a member of the miRNAs family, miR-126can regulated the cell apoptosis in mouse artery atherosclerosis model. The role is down regulator for the formation of blood vessels by the inhibition of cell-derived factor1expression in human endothelial cells. Regulation of vascular integrity of the vascular endothelial cell proliferation and angiogenesis. Sun X, and other studies suggest that the downward and upward role of miR-126was not no significant correlation with coronary heart disease. It had only significant correlation with cholesterol metabolism and the expression levels in the low-density cholesterol (LDL) in patients with high coronary heart disease was reduced. Guangwen Long thought that miR-126expression was correlation with acute myocardial infarction and the expression of miR-126in patients with acute myocardial infarction is reduced. He found the up and down regulation was no correlation with time. In different expression of time curve curve is consistent with the expression and serum troponin I, but no significant correlation with each other.MiR-92a as a another member of the miRNAs family.The study found miR-92a was highly expressed in the control of angiogenesis in human endothelial cells. Overexpression of miR-92a in endothelial cells in vivo and in vitro would damaged angiogenesis. In vitro studies have shown that inducible ischemia miR-92a expression was significantly up-regulated in the organization. Inhibition of miR-92a expression in acute myocardial infarction model could promote the recovery of heart function. Experimental studies have shown that miR-92a was expressed not only in the endothelial cells and also in cardiac fibroblasts, cardiac myocytes. Antagonizing miR-92a could be significantly reduced apoptosis. It did not affect cell survival. The study also found that miR-92a dependent regulation of NO synthase to control vascular tone. Studies have shown that miR-92a was high expressed in patients with coronary heart disease. Statins could reduce miR-92a expression to reduce blood lipids. Inhibition of miR-92a might be reduced myocardial infarct size and improved myocardial infarction remodeling and neovascularization. Studies have shown that miR-92a by promoting cardioprotective protein synthesis played a positive role in myocardial ischemia/reperfusion.
Therefore, this study from the clinical and basic aspects of miR-92a and miR-126in myocardial ischemia and reperfusion in the role of research, methods and measures for the diagnosis and treatment of coronary heart disease in the future.
Objective:
1. Could the detection of miR-92a and miR-126be the early diagnosis of acute myocardial infarction by the miR-92a and miR-126expression studies in the peripheral blood of patients with acute myocardial infarction?
2. To elaborate the possible role of the miRNAs in reperfusion of acute myocardial infarction in patients by detecting the miR-92a and miR-126expression in peripheral blood before and after treatment of reperfusion
3. To explain the possible role of miR-92a and miR-126in the no-reflow phenomenon after reperfusion therapy for acute myocardial infarction through studying the expression of miR-92a and miR-126.4.To infer the possible mechanism of miR-92a and miR-126in myocardial ischemia reperfusion injury regulatory through the establishment of the mice with myocardial ischemia and reperfusion and the experimental studies.
Methods:
1.The collection and testing of specimens for clinical peripheral blood in miRNAs
1.136patients with coronary heart disease and3health s which age to match the patients were selected into the study of clinical.
Clinical studies. Groups:patients with unstable angina, patients with unstable angina and non-ST-segment elevation acute myocardial infarction, ST-segment elevation myocardial infarction group and the healthy control group. All patients with coronary heart disease were treated with Percutaneous coronary intervention (PCI) treatment and patients with AMI are undergoing emergency PCI treatment.
1.2The venous blood in the patients with coronary heart disease was taken before PCI,1and5days after PCI. So did the healthy control group. Their blood were collected to separate vials for good transferr. Putting it to-70℃freezer in short-term storage and liquid nitrogen for long-term preservation.
1.3Using real-time quantitative fluorescent PCR (qRT-PCR) to measure the expression of miR-92a, miR-126. All detection processing was carried out in accordance with the kit instructions on kangcheng company in shanghai city. At the same time we detected indicators in the blood of patients,such as.:the serum troponin I (cTNI), N-terminal pro-brain natriuretic peptide (NT-proBNP), ultra-sensitivity C-reactive protein (hs-CRP), the DD dimer (D-D). we analysised the patients with a complex flow phenomena after PCI surgery.
1.4Statistical analysis:SPSS13.0statistical software. Using the t test or analysis of variance, the parameter is x±s. The correlation analysis is the Pearson correlation method, P<0.05was considered statistically significant.
2. The collection and testing of specimens for animal experiments peripheral blood in miRNAs
2.1Mice myocardial ischemia-reperfusion model and grouping:The left coronary descending artery of Kunming mice was ligation. The successfully mark of model is the ECG ST-segment elevation and mouse myocardial white colors. Thirty minutes later, removing of the ligature. The mouse myocardial colors recover to red and ST segment depression as a sign of success of reperfusion model of myocardial recovery. This animal model is mature. Groups:sham group (9cases), model group (9cases), the the intervention model group (9cases), with three in each group were made to histological sections. The intervention model group was given Atorvastatin (2mg/kg) and the other two groups were given saline, a total of seven days.
2.2Specimen collection and completion of relevant indicators
Six mouse in each group were designed to coronary artery ligation for30minutes before reperfusion12h. Some portion of the serum blood was used to detect the related indicators,the other whole blood (about400ul) were collected good transferred to separate vials putting to-70℃refrigerator for short-term storage and liquid nitrogen for long-term preservation. After killed mice different specimens (ischemia area, marginal zone) organization were deal with by fixed-dehydration-embedded in paraffin-slices.
2.3To detect relevant indicators
The specimens was stained by Evans blue. The apoptotic index was detected by the TUNEL method. Whole blood was used by quantitative real-time fluorescence PCR (qRT-PCR) to determine the expression of miR-92a, miR-126. CK-MB with immune chemiluminescence, a chemical test of superoxide dismutase (SOD), malonic aldehyde (MDA).
2.4statistical analysis
SPSS13.0statistical software. Using the t test or analysis of variance, the parameter is x±s. The correlation analysis is the PERSON correlation method, P<0.05was considered statistically significant.
Results:
Results of clinical trials
1.The results of qRT-PCR showed that the expression level of miR-92a and the healthy control group of patients with coronary heart disease is increased between the two group.There is a significant difference (P<0.05); In the different types of patients with coronary heart disease the miR-92a expression increased with the severity of increased (P<0.05); After PCI miR-92a expression levels reduced.The miR-92a expression levels between before and after PCI was significant differences (P<0.05); same type of patients with coronary heart disease the expression level of miR-92a reduced over the time change (P<0.05).
2. The expression level of miR-126and the healthy control group of patients with acute myocardial infarction (non-ST-segment elevation myocardial infarction and ST-segment elevation myocardial infarction) reduced, a significant difference between the two group (P<0.05); MiR-126expression levels of other types of patients with coronary heart disease and the healthy control group had no significant difference (P>0.05); Acute myocardial infarction after PCI miR-126expression levels increased. The miR-126expression between before and after PCI was significant differences (P<0.05);The same type of patients miR-126expression levels with coronary heart disease after PCI increased and changed over time (P <0.05).
3we found that the cTNI,hs-CRP and DD.level in the blood of patients with acute myocardial infarction were not correlated with the expression level of miR-92a was (r=0.162,0.424,0.361,P>0.05). NT-proBNP levels was significantly correlated with miR-92a expression levels (r=0.583, P<0.05). NT-proBNP, hs-CRP, DD levels were significantly correlated with miR-126expression levels (r were-0.605,-0.666,-0.616, P<0.05). cTNI were not significant correlation with the expression level of miR-126(r=-0.368, P>0.05).
4. The study about no-reflow phenomenon in patients with acute myocardial infarction and reperfusion treatment of the emergence PCI:(1) miR-126expression levels, miR-92a expression levels, hs-CRP levels and D-D levels between the two groups was significant difference (P<0.05).(2) miR-126expression levels was significantly correlated with hs-CRP, D-D levels (r1=-0.666, r2=-0.616, P<0.05), there are significant relationship between the expression level of miR-92a and hs-CRP levels, D-D level (r1=0.638, r2=0.623, P<0.05).
The results of animal experiments
1.The miR-92a, miR-126relative expression level (2-△△CT)were detected by using qRT-PCR method in the mice myocardial ischemia-reperfusion group (sham group, ischemia-reperfusion group, drug intervention group).We found that the expression levels both in the sham group and ischemia-reperfusion group were significant difference in the drug intervention group (P<0.05)
2. The relationship between serum markers of myocardial cell apoptosis index and miR-92a and miR-126in three groups:Sham group, ischemia-reperfusion group, drug intervention reperfusion group.(1) miR-92a and CK-MB, MDA changes are consistent with and the change of SOD is the opposite.(2) The change of miR-126expression is opposite to the changes of CK-MB and MDA and are consistent with SOD.(3) The changes miR-92a expression is consistent apoptosis index trend. The trend of changes in the amount of miR-126expression is opposite to apoptosis index.
Conclusions:
1. MiR-92a and miR-126were involved in the process of myocardial ischemia and reperfusion. It is Possible for early diagnosis and prediction of no-reflow phenomenon and may provide ideas for early intervention.
2.We fond that the expression of miR-92a and miR-126was associated with oxygen free radicals and myocardial apoptosis in myocardial ischemia-reperfusion injury,
3.The intervention of statins can alter the expression of miR-92a and miR-126.
冠心病是导致人类死亡的主要病因之一,因为心肌缺血损伤和缺血再灌注损伤导致患者死亡或心力衰竭发生率非常高。对于急性心肌梗死患者,利用溶栓或早期用经皮冠状动脉介入治疗进行有效的心肌再灌注是缩小心肌梗死面积,改善临床转归的有效方法。但是对于心肌缺血后损伤和缺血再灌注损伤对心功能的影响和突发心脏事件的发生如何预测和减低是非常棘手的问题。尽管我们对心肌缺血再灌注损伤的机制目前研究发现主要认为是中性粒细胞聚集、钙超载或钙再分布、线粒体能量合成障碍、氧自由基生成、细胞因子、心肌细胞凋亡等现象所引起的,但是我们是否能对引起这些变化的相关调控物质予以检测和干预来达到理想的效果呢?
缺血是由于供血障碍引起的组织供血不足的病理表现,组织缺血导致的疾病称缺血性疾病,是可以发生在多种组织器官上的常见疾病。再灌注是指组织缺血后恢复供血的过程,缺血再灌注实际上包括缺血和再灌注两个方面,缺血是引起疾病的原因,,也是再灌注的条件。及时的心肌再灌注治疗,如溶栓、经皮冠状动脉介入治疗(PCD等都可以重建冠状动脉血运,使缺血心肌得到再灌注,从而减少心肌坏死面积,是急性心肌梗死(AMI)治疗的关键。但近来研究发现,冠状动脉的骤然开通与血流恢复,会引起血管内皮细胞功能异常、激活相关炎症因子等,反而会加重缺血心肌的损伤,导致再灌注损伤。AMI的动物研究结果已经表明,40%-50%的坏死心肌为再灌注损伤所引起,这种损伤的存在减弱了积极再灌注治疗所得到的益处。因此,减少再灌注损伤成为AMI防治的非常重要环节之一。
心肌缺血后损伤和缺血再灌注损伤目前主要有以下几种观点：1、自由基损伤学说。当机体损伤产生的自由基超过机体的清除能力时,将对机体产生损伤作用。研究证实缺血再灌注过程中,缺血区有大量的自由基产生,如清除氧自由基能力降低或活性氧自由基(ROI)产生过多,都会导致ROI蓄积,从而引发心肌氧化应激。氧化应激是缺血组织再灌注的特征之一。而且研究证实应用自由基清除剂辅酶Q10可以减轻缺血再灌区细胞的损伤。2、钙超载是造成心肌细胞损伤的重要机制之一,缺血缺氧时心肌细胞内酸中毒,细胞内H+增加,恢复再灌注时细胞内外形成pH梯度差,刺激Na+-H+交换,使Na+大量内流。再灌注后由于能量供应和pH值的恢复,促进Na+-Ca2+交换,,细胞外的Ca2+大量内流,,造成Ca2+超载。从而使细胞膜和细胞器膜受损、促进氧自由基生成而导致心肌损伤。同时由于缺血时血管平滑肌有明显Ca2+内流增加,可致血管收缩痉挛,血管阻力增加对缺血区循环不利,使心肌梗死灶扩大。研究发现,细胞内钙离子浓度的改变是造成再灌注损伤的重要原因之一,钙超载还是多种原因导致的细胞损伤和死亡的共同通路。3、心肌能量代谢障碍。三磷酸腺苷(ATP)是细胞代谢的主要能量来源,细胞所处的生命状态和一切生命活动最终都依赖AT P的水平。如果轻度缺氧或短暂严重缺氧后复氧,细胞仍可生成部分ATP,可为凋亡所需能量提供保障,促使细胞发生凋亡。反之,如果较长时间严重缺氧,细胞由于AT P骤减导致凋亡所需能量不足而发生坏死；当ATP生成被完全抑制后细胞发生坏死,提示ATP在缺氧诱导细胞死亡过程中是细胞死亡方式的决定性因素之一。能量代谢障碍也是自由基产生的基础,自由基损伤又可加重能量代谢障碍,两者也是互为因果的关系。所以说,线粒体损伤和能量代谢障碍是心肌缺血再灌注损伤的重要原因,细胞内的AT P水平是决定细胞发生凋亡或坏死的主要因素。4、内皮细胞抗氧化系统损伤,研究证明内皮细胞的抗氧化活性与其对氧化活性的敏感性之间有一定的关系。心肌缺血再灌注时,内皮细胞不仅产生大量的活性氧,而且其抗氧化活性大大降低,并对外源性的活性氧产生系统有较高的敏感性,从而引起大量活性氧的产生,远远超过了内皮细胞的防御系统,最终引起心肌的损伤。有研究表明,缺血再灌注发生的内皮细胞功能障碍及NO合成的减少,内皮素(ET)心肌缺血时ET释放增加,再灌注时ET释放进一步增加,引起了强烈的血管收缩,可直接引起心肌缺血,加重血管功能障碍,使细胞发生不可逆的损伤。组织缺氧及肾上腺素分泌增加均可导致ET的mRNA表达及ET释放,心肌缺血再灌注还可引起心肌细胞膜ET受体上调,并使冠状血管对ET的敏感性增加,冠脉小分支因而易于痉挛,可导致心肌的无复流现象。内皮细胞与中性粒细胞粘附缺血、再灌注引起中性粒细胞与内皮细胞的粘附增加,中性粒细胞与血管内皮接触时即被激活,释放OFR等毒性产物及破坏性蛋白酶,改变血管的通透性。同时活性氧产生的增加,损伤了内皮细胞和其他细胞,其作用于内皮细胞或粒细胞表面的粘附分子,促进内皮细胞的粘附,而缺氧本身也损伤内皮功能,改变其粘附性,最终导致内皮细胞破损、水肿和功能障碍,毛细血管腔被阻塞,导致虽有大血管的再灌注但局部缺血区仍无复流的现象。5、相关细胞因子作用。由于缺氧复氧的刺激,冠状血管受损,这时肿胀的内皮细胞阻碍了气体交换,而内皮细胞和平滑肌细胞的损伤使血管不能很好扩张,血管腔内中性粒细胞和血小板的聚集通过嵌顿、堵塞毛细血管,使冠脉血流更趋减少；继而血管内皮和中性粒细胞产生细胞因子、粘附因子,使中性粒细胞向血管内皮细胞粘附,并释放颗粒状弹性硬蛋白、活性氧、溶酶体酶、细胞因子和其他炎性介质。这些物质会损伤内皮、血管平滑肌细胞和心肌细胞。6、细胞凋亡参与了这一过程,细胞凋亡是缺血再灌注组织损伤功能丧失的重要原因,它是一个很复杂的过程。细胞凋亡的诱导因素很多,而在心肌缺血再灌注引起的细胞凋亡中主要机制则为自由基增多和细胞内Ca2+水平升高。由于缺血缺氧可使内源性抗氧化剂如SOD失活或耗尽,从而使AT P代谢产物在缺血缺氧心肌组织中堆积,产生大量氧自由基。氧自由基性质极不稳定,一旦形成迅速使肌膜的葡萄糖与脂质过氧化,蛋白质变性,酶失活,并使细胞内的DNA链断裂,从而诱导细胞凋亡。
目前对于微小RNA在心血管系统的作用研究处于热点中。微小RNA(microRNA, miRNA)是近年发现的小的、内源性的单链的非编码RNA,大约由22个核苷酸组成,与基因表达的控制有关。它被认为是基因表达的负面调节器,通过与其目标mRNA分子的3’端非编码区互补匹配导致该mRNA分子的翻译受到抑制。目前大约有三分之一的基因受miRNA调控。最近的研究表明一些miRNA在心血管高度表达,它们在调控心血管的发育和疾病方面具有重要意义,这给心血管疾病的治疗带来了希望。研究发现大鼠离体和活体的心肌缺血/再灌注模型中miR-320的表达式持续性失调。鉴定热休克蛋白20(一种已知的心肌保护蛋白)是miR-320的靶体。敲除内源性miR-320发现通过热休克蛋白20来保护有心肌缺血再灌注诱导的心肌细胞死亡/凋亡将失去。另外的研究发现大鼠缺血6小时后心脏的缺血区miR-21的表达是明显下降的,而在周围边缘区表达是上升的。在离体转染miR-21通过其靶基因执行激活蛋白1通路来降低边缘区和缺血区细胞的凋亡。在活体中实验表明miR-1和miR-133对细胞的凋亡是起相反作用的。miR-1还通过抑制胰岛素样生长因子1的转运与细胞的死亡是联系在一起的。最近的研究发现在病人的外周循环血中也可检测出miRNAS。因此,我们可以假设外周血中miRNAs反映了组织的损害,基于此miRNAs可以作为急性心肌梗死的血清生物标记物。
miR-126(microRNA-126)作为miRNAs家族中的一员,人们通过对小鼠动脉粥样硬化模型的研究,发现其在其中有调节细胞凋亡的作用。在人的内皮细胞中,对血管的形成起副调节作用,抑制细胞衍生因子1的表达。调节血管的完整性、血管内皮细胞的增值和新生血管的形成。Sun X等研究认为miR-126的下调和上调和冠心病发病无明显相关,只是和胆固醇的代谢有相关性,在高的冠心病伴低密度胆固醇(LDL)的患者中的表达量是减少的。但是Guangwen Long等在急性心肌梗死中测得血浆中的miR-126表达和急性心肌梗死有关,在急性心肌梗死中的miR-126表达是减少的,且其上调和下调与时间相关,其在不同时间的表达曲线基本是和血清肌钙蛋白Ⅰ的时间表达曲线是一致的,但彼此未见明显相关性。miR-92a作为miRNAs家族中的另一员,研究发现在人的内皮细胞中高度表达,控制新的血管生成。体内和体外的研究发现miR-92a在内皮细胞中的过度表达损害血管生成。离体的研究表明诱导缺血后组织中的miR-92a的表达明显上调。在急性心肌梗死模型中抑制miR-92a的表达回促进心脏功能的恢复。实验研究表明miR-92a不仅在内皮细胞中表达,而且在心脏成纤维细胞、心肌细胞中,拮抗niR-92a能明显降低细胞凋亡,但并没有影响细胞的生存。研究还发现miR-92a依赖性调节NO合酶来控制血管张力。研究表明在冠心病患者中的表达偏高,他汀类可以通过减低miR-92a表达来减低血脂。抑制了miR-92a可能会减少心肌梗死面积,改善心肌梗死后重塑和新生血管。在心肌缺血／再灌注中研究表明miR-92a通过促进心脏保护蛋白的合成,起到积极作用。因此,本研究从临床和基础方面对miR-92a和miR-126在心肌缺血再灌注中作用进行研究,为未来冠心病的诊治提供方法和措施。
研究目的：
1、通过对急性心肌梗死患者外周血中的miR-92a和miR-126表达研究,检测miR-92a和miR-126能否进一步早期诊断急性心肌梗死；
2、对急性心肌梗死患者行再灌注治疗前后外周血中miR-92a和miR-126表达来阐述微小RNA在其中的可能作用。
3、通过对急性心肌梗死行再灌注治疗后出现无复流现象时miR-92a和miR-126表达的研究,试图说明miR-92a和miR-126在其中的可能作用。
3、通过建立小鼠的心肌缺血再灌注实验研究来推断miR-92a和miR-126在心肌缺血再灌注损伤中可能的调控机制。
研究方法：
1、临床外周血中微小RNA等相关标本的收集和检测
1.1选择了36例证实为冠心病患者和与之年龄向匹配的3例健康者进入临床研究。分组：稳定性心绞痛组、不稳定性心绞痛组、非ST段抬高性急性心肌梗死组、ST段抬高性急性心肌梗死组、健康对照组。所有冠心病患者均行PCI治疗,AMI患者行急诊PCI治疗。
1.2取冠心病患者PCI术前和PCI术后1、5天的静脉血。健康对照组为匹配的时间。将采集好的全血转移至单独的冻存管,短期保存,放入-70℃冰箱,长期保存,放入液氮中。
1.3采用实时定量荧光PCR (qRT-PCR)来测定miR-92a、miR-126的表达,所有检测过程按照上海康成公司提供的试剂盒说明来进行。同时检测患者血中的相关指标：清肌钙蛋白I (cTNI)、N-端脑钠肽前体(NT-proBNP)、超敏C反应蛋白(hs-CRP)、D-D二聚体(D-D)等指标。并分析PCI手术时患者是否有无复流现象。
1.4统计学分析：采用SPSS18.0统计软件。使用t检验或方差分析,参数以x±S表示。相关分析采用Pearson或Spearman相关分析法,P<0.05为差异有统计学意义
2、动物实验中外周血中微小RNA等相关标本的收集和检测
2.1小鼠心肌缺血再灌注模型的建立及分组：
选昆明小鼠结扎其左冠状动脉前降支,以小鼠心肌颜色变白且心电图ST段持续弓背抬高为模型成功标志。结扎30分钟后,撤除结扎线,以心肌恢复红润且ST段下降作为再灌注模型成功标志。目前此动物模型建立是成熟的。分组：假手术组(9只)、模型组(9只)、干预模型组(9只),每组中的3只用制成组织学切片。干预模型组用阿托伐他汀(2mg/kg)每天一次灌胃,其他两组给予等量的生理盐水,共7天。
2.2标本采集和相关指标的完成
各组小鼠分别设计为结扎冠状动脉30分钟后再灌注12h各6只,取部分血用来检测血清中相关指标,另取血400ul,将采集好的全血转移至单独的冻存管,短期保存,放入-70℃冰箱,长期保存,放入液氮中。杀死小鼠后取不同标本(缺血区、边缘区)组织经固定-脱水-石蜡包埋-切片。
2.3检测相关指标
用伊文氏蓝来进行染色。细胞凋亡指数用TUNEL法。全血采用实时定量荧光PCR (qRT-PCR)来测定miR-92a、miR-126的表达。CK-MB用免疫发光法,用化学法测试超氧化物歧化酶(SOD)、丙二醛(MDA)。
2.4统计学分析
采用SPSS13.0统计软件。使用t检验或方差分析,参数以x±S表示。相关分析采用Pearson或Spearman相关分析法,P<0.05为差异有统计学意义
结果：
临床试验的结果
1、qRT-PCR显示,冠心病患者的]miR-92a表达量与健康对照组是增加的,两者之间有显著性差异(P<0.05)；不同类型冠心病患者miR-92a表达量随病情程度加重而升高(P<0.05); PCI术后miR-92a表达量是降低的,PCI术前后miR-92a表达量有显著性差异(P<0.05)；同一类型冠心病患者PCI术后随时间的变化miR-92a表达量降低(P<0.05)。
2、急性心肌梗死患者(包括非ST段抬高性心肌梗死和ST段抬高性急性心肌梗死)的miR-126表达量与健康对照组是降低的,两者之间有显著性差异(P<0.05)；其他类型冠心病患者miR-126表达量与健康对照组无显著性差异(P>0.05)；急性心肌梗死PCI术后miR-126表达量是增加的,PCI术前后miR-126表达量有显著性差异(P<0.05)；同一类型冠心病患者PCI术后随时间的变化miR-126表达量增加(P<0.05)。
3、急性心肌梗死患者血中的血清肌钙蛋白I (cTNI)、超敏C反应蛋白(hs-CRP)、D-D二聚体(D-D)的水平,发现这些指标均与miR-92a表达量无统计学意义的相关关系(r=0.162, P=0.515; r=0.424, P=0.079; r=0.361, P=0.141)。N-端脑钠肽前体(NT-proBNP)与miR-92a表达量有统计学意义的相关关系(r=0.583,P=0.011)。N-端脑钠肽前体(NT-proBNP)、超敏C反应蛋白(hs-CRP)、D-D二聚体(D-D)的水平均与miR-126表达量有统计学意义的相关关系(r=-0.605, P=0.033; r=-0.666, P=0.008; r=-0.616, P=0.008)。血清肌钙蛋白I (cTNI)与miR-126表达量无统计学意义的相关关系(r=-0.368,P=0.133)。
4、急性心肌梗死患者再灌注治疗出现无复流现象分析：(1)miR-126表达量、miR-92a表达量、hs-CRP水平、D-D水平在两组之间有显著性差异(P<0.05)。(2)miR-126表达量与hs-CRP水平、D-D水平有统计学意义的相关关系(r1=-0.666,r2=-0.616,P<0.05),miR-92a表达量与hs-CRP水平、D-D水平有统计学意义的相关关系(r1=0.638,r2=0.623,P<0.05)。
动物实验的结果
1、通过使用qRT-PCR法检测出的小鼠心肌缺血再灌注不同组别(假手术组、缺血再灌注组、药物干预再灌注组)中全血中的：miR-92a.miR-126相对表达量(2-△△CT),经过分析：两者在假手术组、缺血再灌注组、药物干预组的表达量有显著性差异(P<0.05)
2、假手术组、缺血再灌注组、药物干预再灌注组三组中miR-92a和miR-126和血清标志物及心肌细胞凋亡指数之间的相关关系：(1)miR-92a和CK-MB、MDA的变化是一致,而与SOD的变化是相向的。(2)miR-126表达量的变化和CK-MB、MDA的变化是相反,而与SOD的变化是一致的。(3)miR-92a表达量变化心肌细胞凋亡指数的变化趋势是一致的。而miR-126表达量的变化和心肌细胞凋亡指数的变化趋势是相反的。
结论：
1、发现miR-92a和miR-126参与了心肌缺血再灌注的过程。可能为早期诊断和预测无复流现象及可能提前干预提供思路。
2、在心肌缺血再灌注损伤中,发现miR-92a和miR-126的表达与氧自由基和心肌细胞凋亡有关联。
3、他汀类药物的干预可以改变miR-92a和miR-126的表达。
Background
Coronary heart disease is one of the major reasons of human death, because myocardial ischemic injury and ischemia-reperfusion Injury can lead to death or very high incidence of heart failure. For patients with acute myocardial infarction, the using of thrombolysis or percutaneous coronary intervention in the early treatment can reduce myocardial infarct size after the myocardial reperfusion and can be an effective way to improve clinical outcome. However, it is very difficult how to predict and reduce damage after myocardial ischemia and ischemia-reperfusion injury of cardiac function and sudden cardiac events. By studying the mechanism about myocardial ischemia and reperfusion injury, we found that the main reasons are neutrophil accumulation, calcium overload or calcium redistribution, of mitochondrial energy synthesis barriers,the generation of oxygen free radicals. But can we achieve the desired effect by studying these changes with regulators detection and intervention? Recently it is the hotspot research to study the role of microRNA in the cardiovascular system.
Ischemia is a blood disorder caused by insufficient blood supply to tissue pathology. Tissue ischemia caused by the disease known as ischemic disease, it can happen in a variety of tissues and organs of the common diseases.
Reperfusion after ischemia refers to the organization of the process to restore the blood supply.
Ischemia-reperfusion actually includes two aspects.One is ischemia and another is reperfusion. The cause of the disease is not only caused by ischemia, but also the conditions of reperfusion. Timely reperfusion therapy, such as thrombolysis, percutaneous coronary intervention (PCI), etc. can be reconstructed coronary revascularization, myocardial reperfusion obtained. Thereby.reducing the area of myocardial necrosi is the key way of the treatment about acute myocardial infarction (AMI). However, recent studies have found that a sudden opening of the coronary arteries and restore blood flow might cause endothelial cell dysfunction, activation of inflammatory factors. But increasing ischemic myocardial injury could result in reperfusion injury. Animal with AM study results I show that40%to50%of myocardial necrosis is caused by reperfusion injury and this injury weakens the active presence of reperfusion therapy benefits obtained. Therefore, it is to reduce reperfusion injury that become an important part of prevention and treatment of AMI.
Injury after myocardial ischemia and ischemia-reperfusion injury is currently mainly the following points:1, free radical damage theory. When the body produces free radicals damage exceeding the body's ability to clear, the body will produce injury. Study confirmed during ischemia-reperfusion, ischemic area there are plenty of free radicals, such as reduced ability to scavenge oxygen free radicals or reactive oxygen species (ROI). ROI can produce too much will lead to ROI accumulation caused by oxidative stress. Oxidative stress reperfusion of ischemic tissue is one of the characteristics. And the application of free radical scavengers Coenzyme Q10can reduce ischemic cell damage irrigation.
2, Calcium overload caused by myocardial injury is an important mechanism of ischemic hypoxia.The myocardial intracellular acidosis, intracellular H+to increase and restore reperfusion intracellular formation of pH gradient difference, stimulating Na+-H+exchange, so that Na+a large influx. Since the energy supply after reperfusion and pH recovery, promote Na+-Ca2+exchange, a large number of extracellular Ca2+influx, resulting in Ca2+overload. So that the cell membrane and organelle membrane damage caused by oxygen free radicals promote myocardial injury. Ischemic vascular smooth muscle and because a significant increase in Ca2+influx can cause vasoconstriction spasm, vascular resistance increased circulating adverse ischemic area, expand the infarct. Study found that the changes of the concentration of intracellular calcium are caused by reperfusion injury.It is one of the important reasons. Calcium overload is a variety of causes cell damage and death common pathway.
3,Myocardial energy is metabolism, ATP is the main energy source of cell metabolism, cell in which the life of the state and all life activities ultimately depend AT P levels. If mild or transient hypoxia reoxygenation after severe hypoxia, cells can still generate some ATP and the energy required for apoptosis to provide protection to promote apoptosis. Conversely, if a longer time severe hypoxia,cell apoptosis due to AT P plummeted lead to insufficient energy needed necrosis; When ATP production was completely inhibited cell necrosis, Study suggests that hypoxia-induced ATP in the cell during cell death manner of death of the decisive factors. Energy metabolism is the basis of free radicals, free radicals can also increase energy metabolism, they are mutually reinforcing relationship. Therefore, mitochondrial damage and energy metabolism of myocardial ischemia-reperfusion injury is a major cause of intracellular AT P. It is to determine the level of apoptosis or necrosis of the main factors.
4,Anti-oxidation system damage endothelial cells. Studies have shown that the antioxidant activity of endothelial cells and their sensitivity to oxidation activity relationship between certain. When myocardial ischemia and reperfusion, the endothelial cells not only produce large amounts of reactive oxygen species, and also greatly reduce its antioxidant activity.Exogenous reactive oxygen generation system has high sensitivity. It can cause a large number of reactive oxygen species, far endothelial cells than defense system, and ultimately lead to myocardial injury. Other studies have shown that ischemia-reperfusion endothelial cell dysfunction occurs and the reduction of NO synthesis,. Endothelin (ET) ET increase release of myocardial ischemia. Reperfusion ET release further increased causing intense vasoconstriction.It can be directly cause myocardial ischemia, increased vascular dysfunction, cell irreversible damage. Tissue hypoxia and increased secretion of adrenaline can lead to the ET mRNA and ET releasing. Myocardial ischemia and reperfusion can cause myocardial cell ET receptors, and makes the ET coronary vessels increased sensitivity to small branches. And thus coronary Easy spasms that can lead to myocardial no-reflow phenomenon. Endothelial cells and neutrophil adhesion ischemia, reperfusion induced neutrophil adhesion to endothelial cells increased neutrophils and endothelial contact is activated, the release of OFR and other toxic products and destructive proteases, vascular permeability changes. At the same time an increasing in reactive oxygen species will damage to the endothelial cells and other cells, and its role in endothelial cells or granulocyte cell surface adhesion molecules, promote endothelial cell adhesion and endothelial function hypoxic injury itself. Changing its adhesion eventually leading to endothelial cell damage, edema and dysfunction. Capillary chamber is blocked, leading to the great vessels although the area of ischemia-reperfusion, but still no reflow phenomenon.
5, the related cytokines. Bing stimulation of the hypoxia when hindered gas exchange, coronary vascular will injury swelling of endothelial cells. And endothelial cells and smooth muscle cell damage the blood vessels are not well expanding the endovascular neutrophil and platelet aggregation through incarceration, clogging the capillaries, so that more reduced coronary blood flow;Followed by vascular endothelium and neutrophils produce cytokines, adhesion molecules, the neutrophils to vascular endothelial cells stick attached, and the release of particulate elastin, reactive oxygen species, lysosomal enzymes, cytokines and other inflammatory mediators. These substances can damage the endothelium, vascular smooth muscle cells and cardiac myocytes.
6.Involving in the process of cell apoptosis. The apoptosis is ischemia-reperfusion tissue injury in important cause loss of function. It is a very complicated process. Cell apoptosis is Inducted by many factors. And in myocardial ischemia and reperfusion inducing cell apoptosis was the main mechanism.Intracellular free radicals increased Ca2+levels. Because ischemic hypoxia endogenous antioxidants such as SOD inactivation or depletion, AT P metabolites accumulate in hypoxic-ischemic myocardial tissu eand result in a large number of oxygen free radicals. Unstable nature of oxygen free radicals once formed to intracellular DNA strand breaks quickly make sarcolemma glucose and lipid peroxidation, protein denaturation and enzyme inactivation, and can induce apoptosis.
MicroRNAs is a recently discovered small endogenous non-coding single-stranded RNA(about22nucleotides) and the control of gene expression. It is considered to be a negative regulator of gene expression, via its target mRNA molecule3'end complementary to the non-coding region match by inhibiting translation of the mRNA molecule (1-3). Currently, there is about one-third of the genes regulated by MicroRNAs. Recent research suggests that some MicroRNAs are highly expressed in the cardiovascular and they are of great significance in the regulation of cardiovascular development and disease, which will bring hope to the treatment of cardiovascular diseases. The study found that the persistent disorders of miR-320expression in model of the rats away from the body and in vivo myocardial ischemia/reperfusion.They identify that heat shock protein20(a known myocardial protection protein) is the target body of miR-320. After knockdown of endogenous miR-320, the heat shock protein20will lose the protection myocardial ischemia-reperfusion-induced myocardial cell death/apoptosis. Another study found that the miR-21Expression of the ischemic area of the heart is decreased after six hours of ischemia in rats, while the expression is increased in the peripheral edge area. Transfected with miR-21in vitro through the implementation of its target gene activator protein1pathway to reduce cell apoptosis in the edge of the area and the ischemic area. In the vivo experiments of MiR-1and miR-133show that the effect of apoptosis is counterproductive. MiR-1is linked to cell death by inhibiting the transporter of insulin-like growth factor1. Recently study found that miRNAs can be detected in the blood of the patient's peripheral circulation. Therefore, we can assume that the peripheral blood miRNAs reflect damage of tissue. So the detected miRNAs can be serum biomarkers as acute myocardial infarction,.
As a member of the miRNAs family, miR-126can regulated the cell apoptosis in mouse artery atherosclerosis model. The role is down regulator for the formation of blood vessels by the inhibition of cell-derived factor1expression in human endothelial cells. Regulation of vascular integrity of the vascular endothelial cell proliferation and angiogenesis. Sun X, and other studies suggest that the downward and upward role of miR-126was not no significant correlation with coronary heart disease. It had only significant correlation with cholesterol metabolism and the expression levels in the low-density cholesterol (LDL) in patients with high coronary heart disease was reduced. Guangwen Long thought that miR-126expression was correlation with acute myocardial infarction and the expression of miR-126in patients with acute myocardial infarction is reduced. He found the up and down regulation was no correlation with time. In different expression of time curve curve is consistent with the expression and serum troponin I, but no significant correlation with each other.MiR-92a as a another member of the miRNAs family.The study found miR-92a was highly expressed in the control of angiogenesis in human endothelial cells. Overexpression of miR-92a in endothelial cells in vivo and in vitro would damaged angiogenesis. In vitro studies have shown that inducible ischemia miR-92a expression was significantly up-regulated in the organization. Inhibition of miR-92a expression in acute myocardial infarction model could promote the recovery of heart function. Experimental studies have shown that miR-92a was expressed not only in the endothelial cells and also in cardiac fibroblasts, cardiac myocytes. Antagonizing miR-92a could be significantly reduced apoptosis. It did not affect cell survival. The study also found that miR-92a dependent regulation of NO synthase to control vascular tone. Studies have shown that miR-92a was high expressed in patients with coronary heart disease. Statins could reduce miR-92a expression to reduce blood lipids. Inhibition of miR-92a might be reduced myocardial infarct size and improved myocardial infarction remodeling and neovascularization. Studies have shown that miR-92a by promoting cardioprotective protein synthesis played a positive role in myocardial ischemia/reperfusion.
Therefore, this study from the clinical and basic aspects of miR-92a and miR-126in myocardial ischemia and reperfusion in the role of research, methods and measures for the diagnosis and treatment of coronary heart disease in the future.
Objective:
1. Could the detection of miR-92a and miR-126be the early diagnosis of acute myocardial infarction by the miR-92a and miR-126expression studies in the peripheral blood of patients with acute myocardial infarction?
2. To elaborate the possible role of the miRNAs in reperfusion of acute myocardial infarction in patients by detecting the miR-92a and miR-126expression in peripheral blood before and after treatment of reperfusion
3. To explain the possible role of miR-92a and miR-126in the no-reflow phenomenon after reperfusion therapy for acute myocardial infarction through studying the expression of miR-92a and miR-126.4.To infer the possible mechanism of miR-92a and miR-126in myocardial ischemia reperfusion injury regulatory through the establishment of the mice with myocardial ischemia and reperfusion and the experimental studies.
Methods:
1.The collection and testing of specimens for clinical peripheral blood in miRNAs
1.136patients with coronary heart disease and3health s which age to match the patients were selected into the study of clinical.
Clinical studies. Groups:patients with unstable angina, patients with unstable angina and non-ST-segment elevation acute myocardial infarction, ST-segment elevation myocardial infarction group and the healthy control group. All patients with coronary heart disease were treated with Percutaneous coronary intervention (PCI) treatment and patients with AMI are undergoing emergency PCI treatment.
1.2The venous blood in the patients with coronary heart disease was taken before PCI,1and5days after PCI. So did the healthy control group. Their blood were collected to separate vials for good transferr. Putting it to-70℃freezer in short-term storage and liquid nitrogen for long-term preservation.
1.3Using real-time quantitative fluorescent PCR (qRT-PCR) to measure the expression of miR-92a, miR-126. All detection processing was carried out in accordance with the kit instructions on kangcheng company in shanghai city. At the same time we detected indicators in the blood of patients,such as.:the serum troponin I (cTNI), N-terminal pro-brain natriuretic peptide (NT-proBNP), ultra-sensitivity C-reactive protein (hs-CRP), the DD dimer (D-D). we analysised the patients with a complex flow phenomena after PCI surgery.
1.4Statistical analysis:SPSS13.0statistical software. Using the t test or analysis of variance, the parameter is x±s. The correlation analysis is the Pearson correlation method, P<0.05was considered statistically significant.
2. The collection and testing of specimens for animal experiments peripheral blood in miRNAs
2.1Mice myocardial ischemia-reperfusion model and grouping:The left coronary descending artery of Kunming mice was ligation. The successfully mark of model is the ECG ST-segment elevation and mouse myocardial white colors. Thirty minutes later, removing of the ligature. The mouse myocardial colors recover to red and ST segment depression as a sign of success of reperfusion model of myocardial recovery. This animal model is mature. Groups:sham group (9cases), model group (9cases), the the intervention model group (9cases), with three in each group were made to histological sections. The intervention model group was given Atorvastatin (2mg/kg) and the other two groups were given saline, a total of seven days.
2.2Specimen collection and completion of relevant indicators
Six mouse in each group were designed to coronary artery ligation for30minutes before reperfusion12h. Some portion of the serum blood was used to detect the related indicators,the other whole blood (about400ul) were collected good transferred to separate vials putting to-70℃refrigerator for short-term storage and liquid nitrogen for long-term preservation. After killed mice different specimens (ischemia area, marginal zone) organization were deal with by fixed-dehydration-embedded in paraffin-slices.
2.3To detect relevant indicators
The specimens was stained by Evans blue. The apoptotic index was detected by the TUNEL method. Whole blood was used by quantitative real-time fluorescence PCR (qRT-PCR) to determine the expression of miR-92a, miR-126. CK-MB with immune chemiluminescence, a chemical test of superoxide dismutase (SOD), malonic aldehyde (MDA).
2.4statistical analysis
SPSS13.0statistical software. Using the t test or analysis of variance, the parameter is x±s. The correlation analysis is the PERSON correlation method, P<0.05was considered statistically significant.
Results:
Results of clinical trials
1.The results of qRT-PCR showed that the expression level of miR-92a and the healthy control group of patients with coronary heart disease is increased between the two group.There is a significant difference (P<0.05); In the different types of patients with coronary heart disease the miR-92a expression increased with the severity of increased (P<0.05); After PCI miR-92a expression levels reduced.The miR-92a expression levels between before and after PCI was significant differences (P<0.05); same type of patients with coronary heart disease the expression level of miR-92a reduced over the time change (P<0.05).
2. The expression level of miR-126and the healthy control group of patients with acute myocardial infarction (non-ST-segment elevation myocardial infarction and ST-segment elevation myocardial infarction) reduced, a significant difference between the two group (P<0.05); MiR-126expression levels of other types of patients with coronary heart disease and the healthy control group had no significant difference (P>0.05); Acute myocardial infarction after PCI miR-126expression levels increased. The miR-126expression between before and after PCI was significant differences (P<0.05);The same type of patients miR-126expression levels with coronary heart disease after PCI increased and changed over time (P <0.05).
3we found that the cTNI,hs-CRP and DD.level in the blood of patients with acute myocardial infarction were not correlated with the expression level of miR-92a was (r=0.162,0.424,0.361,P>0.05). NT-proBNP levels was significantly correlated with miR-92a expression levels (r=0.583, P<0.05). NT-proBNP, hs-CRP, DD levels were significantly correlated with miR-126expression levels (r were-0.605,-0.666,-0.616, P<0.05). cTNI were not significant correlation with the expression level of miR-126(r=-0.368, P>0.05).
4. The study about no-reflow phenomenon in patients with acute myocardial infarction and reperfusion treatment of the emergence PCI:(1) miR-126expression levels, miR-92a expression levels, hs-CRP levels and D-D levels between the two groups was significant difference (P<0.05).(2) miR-126expression levels was significantly correlated with hs-CRP, D-D levels (r1=-0.666, r2=-0.616, P<0.05), there are significant relationship between the expression level of miR-92a and hs-CRP levels, D-D level (r1=0.638, r2=0.623, P<0.05).
The results of animal experiments
1.The miR-92a, miR-126relative expression level (2-△△CT)were detected by using qRT-PCR method in the mice myocardial ischemia-reperfusion group (sham group, ischemia-reperfusion group, drug intervention group).We found that the expression levels both in the sham group and ischemia-reperfusion group were significant difference in the drug intervention group (P<0.05)
2. The relationship between serum markers of myocardial cell apoptosis index and miR-92a and miR-126in three groups:Sham group, ischemia-reperfusion group, drug intervention reperfusion group.(1) miR-92a and CK-MB, MDA changes are consistent with and the change of SOD is the opposite.(2) The change of miR-126expression is opposite to the changes of CK-MB and MDA and are consistent with SOD.(3) The changes miR-92a expression is consistent apoptosis index trend. The trend of changes in the amount of miR-126expression is opposite to apoptosis index.
Conclusions:
1. MiR-92a and miR-126were involved in the process of myocardial ischemia and reperfusion. It is Possible for early diagnosis and prediction of no-reflow phenomenon and may provide ideas for early intervention.
2.We fond that the expression of miR-92a and miR-126was associated with oxygen free radicals and myocardial apoptosis in myocardial ischemia-reperfusion injury,
3.The intervention of statins can alter the expression of miR-92a and miR-126.
引文
1.王晓良.应用分子药理学[M].北京：中国协和医科大学出版社,2005：215-216.
2. Yellon DM, Hausenloy DJ. Myocardial reperfusion injury [J] NEngl J Med,2007,357(11):1121-1135.
3. ENGLER R L, SCHIMD SCHONBEIN G W, PAVALEC R S.Leu kocyt e capill ary plu gging in m yocar dial isch emia andreperfusion in the dog[J]. Am J Pathol,1983,3:982-987.
4.严勤,苏肇伉,丁文祥.氧自由基清除剂对缺血犬心肌再灌注损伤的保护作用[J].中华胸心血管外科杂志,1995,11(1)：42.
5. ALICIA J K, ROGE R F C, ANIBAL E V. Mit ochond rial p ermeability transit ion and ox idat ive t res s. FEBS let ters [J]. Circ Res,2001,495:12-15.
6. GrossG J, Kersten JR,W arltier DC. Mechan ism s of postisch em ic contractile dysfunct ion[J]. Ann Thorac Su rg,1999,68(5):1898-1904.
7. LOPASCHUK G, ALBERT A E. Regulat ion of carbohydr at emet abolism in i sch emia and reperfusion [J]. Am Heart J,2000,139:S115-S119.
8. HAYASHI H.Pathogen esis and the role of Ca2+ overl oad during myocardial isch emia/reperfus ion [J]. Nagoya J Med Sci,2000,63(3-4):91-98.
9. TE RRY L, VANDEN HOEK, LANC E B, et al. React ive oxygen species released f rom m itochondria du ring b rief hypoxia indu ceprecondit ioning in cardiomyocyt es [J]. J Biol Ch em,1998,273(29):18092-18098.
10. CHUANYU L I, ROBERT M, JAC KSON. React ive s pecies mechanism s of cel lul ar h ypoxia-r eoxygenat ion injury [J]. Am J Physiol Cell Phys iol,2002, 282(2):C227-C241.
11.杨彦玲,师养荣.心肌缺血再灌注损伤研究进展[J].心血管病学进展,2003,24(2)：116-121.
12.梁晚益,黄跃生.线粒体在缺血再灌注细胞损伤中的作用[J].国外医学：生理、病理科学与临床分册,2000,20(5)：364-367.
13. Gotdhab er JI, Q ayyum MSI Oxygen free rad icals and excitat ion con traction coupl ing[J] 1 Ant iox id Redox S igna,l 2000,2(1):55-641
14.吴傅轩,丁文惠,李大元,等1炎症递质在兔心肌缺血再灌注损伤中的作用[J]临床心血管病杂志,2000,16(2)：85-871
15. H am isonWR, Mertis CF, Leathers CR1 Ev idence of collid ividom ycos is in the skeleton of an ancient Ind ia[J]1C irc Res,1997,68:1446-14571
16.丁钢,沈景霞1心肌缺血再灌注损伤中一氧化氮与中性粒细胞之间的关系[J]心血管病学进展,2000,21(5)：290-2921
17.Kiriakidou M, Tan GS, Lamprinaki S, et al. An mRNAm 7G cap binding-like motif within human Ago2 represses translation[J]. Cell.2007,129,1141-1151.
18.Bagga S, Bracht J, Hunter S, et al. Regulation by let-7and lin-4 miRNAs results in target mRNA degradation.Cell.2005,122,553-563.
19.Humphreys DT, Westman BJ, Martin DI et al. MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc Natl Acad Sci USA.2005,102,16961-16966.
20.Lee RC, Feinbaum RL, Ambros V. et al. Elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993;75:843-854.
21.Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell.2005,120,21-24.
22. Pedersen IM, Cheng G, Wieland S, et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature.2007;449:919-922.
23. Kloosterman WP, Lagendijk AK, Ketting RF, et al. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol.2007;5:15-20
24. Tay YM, Tam WL, Ang YS, et al. MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1. Stem Cells.2008;26:17-29.
25.McKinsey TA, Olson EN, et al. Toward transcriptional therapies for the failing heart:chemical screens to modulate genes. J Clin Invest 2005; 115:538-46.
26.van Rooij E, Sutherland LB, Hu N, et al.A signature pattern Of "stress-responsive micmRNAs that can evoke cardiac hypertrophy and heart failure[J]. Proc Nail Aead Sci USA,2006,103(48):18255-18260.
27.Care A, Catalucci D, Felieetti F, et al. MicrORNA-133 controls cardiac hypertrophy[J]. Nat Med,2007,13(5):613-618.
28.Cheng Y, Ji R, Yue J, et al.MieroRNAsmaberrantly expressed in hypertrophic heart:do they play a role in cardiac hypertrophy?[J]. Am J Pathol,2007,170(6): 1831-1840.
29.Tatsuguchi M, Seok HY, Callis TE, et al. Expression of mieroRNAs is dynamically regulated during cardiomyocyte hypertrophy[J]. J Mol Cell Cardiol.2007,42(6):1137-1141.
30. Matkovich SJ, Van Booven DJ, Youker KA, et al. Reciprocal regulation of myocardial microRNAs and messenger RNA in human cardiomyopathy and reversal of the microRNA signature by biomechanical support. Circulation 2009; 119:1263-1271.
31.Schipper ME, van Kuik J, de Jonge N, et al.Changes in regulatory microRNA expression in myocardium of heart failure patients on left ventricular assist device support. J Heart Lung Transplant 2008;27:1282-1285
32.ZhaoY, R,JF, Li A, etal. Dysreguhtion of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-I-2[J]. Cell,2007。129(2):303-317.
33.Yang B, Lin H,Xiao J, et al.The muscle, specific microRNA miR-Ⅰ regulates cardiac arrhythmogenie potential by targeting GJ AI and KCNJ2[J]. Nat Med, 2007,13(4):486-491.
34.Terentyev D, Belevyeh AE, Terentyeva R, etal. MiR-1 overexpres-sion enhances Ca2+release and promotes cardiac arrhythmogenesis by targeting PP2A regulatory subunit B56alpha and causing CaMKII-dependent hyperphesphorylation of RyR2[J]. Cire Res,2009。104(4):514-521.
35.Xiao J,luo X,Lin H. et al. MicroRNA miR-133 represses HERG K+channel expression contributing to QT prolongation in diabetic hearts[J]. J Biol Chem, 2007,282(17):12363-12367.
36.Ono K, Matsumori A, Shioi T, et al. Cytokine gene expression after myocardial infarction in rat hearts:possible implication in left
ventricular remodeling. Circulation.1998,98,149-156.
37.Ren XP, Wu J, Wang X, et al. MicroRNA-320 is involved in the regulation of cardiac ischemia/ reperfusion injury by targeting heat-shock protein 20. Circulation.2009,119,2357-2366.
38.Dong S, Cheng Y, Yang J, et al. MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem.2009,284,29514-29525.
39.Xu C, Lu Y, Pan Z, et al. The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes. J Cell Sci.2007,120,3045-3052.
40.Shan ZX, Lin QX, Fu YH, et al. Upregulated expression of miR-1/miR-206 in a rat model of myocardial infarction. Biochem Biophys Res Commun.2009,381, 597-601.
41.Rabinowits G, Gercel-Taylor C, Day JM et al. Exosomal microRNA:a diagnostic marker for lung cancer. Clin Lung Cancer.2009,10,42-46.
42.Camussi G, Deregibus MC, Bruno S, et al. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int.2010,78,838-848.
43 Ji X, Takahashi R, Hiura Y, et al. Plasma miR-208 as a biomarker of myocardial injury. Clin Chem.2009,55,1944-1949.
44.Wang GK, Zhu JQ, Zhang JT, et al. Circulating microRNA:a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J.2010,31,659-666.
45. D'Alessandra Y, Devanna P, et al. Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J.2010,31,2765-2773.
46.Cheng Y, Tan N, Yang J, et al. A translational study of circulating cell-free microRNA-1 in acute myocardial infarction. Clin Sci (Lond).2010,119,87-95.
47.Ai J, Zhang R, Li Y, et al. Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biochem Biophys Res Commun.2010,391, 73-77.
48.Yu LP, Shi LY, Zhang MM, etal,MicroRNA expression profile and pathogenetic initial study in essential hypertension.Zhonghua Xin Xue Guan Bing Za Zhi.2011 Jun;39(6):488-93. Chinese.
49.Castoldi G, Di Gioia CR, Bombardi C,etal.MiR-133a regulates collagen 1A1: potential role of miR-133a in myocardial fibrosis in angiotensin II-dependent hypertension. J Cell Physiol.2012 Feb;227(2):850-6. doi:10.1002/jcp.22939
50.Wu WH, Hu CP, Chen XP, etal.MicroRNA-130a mediates proliferation of vascular smooth muscle cells in hypertension.Am J Hypertens.2011 Oct;24(10):1087-93.
51.Wang S, Aurora AB, Johnson BA, et al. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell 2008; 15:261-271.
52.van Solingen C, Seghers L, Bijkerk R, et al. ntagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis. J Cell Mol Med 2009;13:1577-1585.
53.Sun X, Zhang M, Sanagawa A, et al. Circulating microRNA-126 in patients with coronary artery disease:correlation with LDL cholesterol.Thromb J.2012 Aug 28;10(1):16.
54.Guangwen Long,* Feng Wang, Quanlu Duan, et al.Human Circulating MicroRNA-1 and MicroRNA-126 as Potential Novel Indicators for Acute Myocardial Infarction Int J Biol Sci.2012; 8(6):811-818. Published online 2012 June 7.
55.Bonauer A, Carmona G, Iwasaki M,et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice[J]. Science 2009,324:1710-3.
56. Wang H, Lu HM, Yang WH et al. Zhong guo Wei Zhong Bing Ji Jiu Yi Xue. The influence of statin therapy on circulating microRNA-92a expression in patients with coronary heart disease [J].2012 Apr,24(4):215-8.
57. Salloum FN, Yin C, Kukreja RC, et al. Role of microRNAs in cardiac preconditioning. J Cardiovasc Pharmacol[J].2010 Dec,56(6):581-8.
58.McKinsey TA, Olson EN. Toward transcriptional therapies for the failing heart: chemical screens to modulate genes[J]. J Clin Invest,2005,115(3):538-546.
59.Steffens S, Montecucco F, Mach F. The inflammatory response as a target to reduce myocardial ischaemia and reperfusion injury[J].Thromb Haemost, 2009,102(2):240-247.
60Ndrepepa G, Braun S, Mehilli J, etal.N-terminal pro-brain natriuretic peptide on admission in patients with acute myocardial infarction and correlation with scintigraphic infarct size, efficacy of reperfusion, and prognosis.Am J Cardiol. 2006 Apr 15;97(8):1151-6. Epub 2006 Mar 3.
61.杨跃进.经皮冠状动脉介入治疗中无再流的诊断、治疗和预防[J].中华心血管病杂志,2004,32(7)：662-664.
62. C arrabba N, Parod i G, Valent i R, et a.l S ign ifican ce of add itional ST segment elevation in patients w ith no reflow afterang iop lasty for acu te myocardial in farct ion. J Am SocE chocardiogr,2007,20:262-269.
63. van d erW alAC, Li X, de Boer O J. T issue factor exp ression in the m orphologic spectrum of vu lnerab le atherosclerotic p laques.Sem in Throm bH emost,2006, 32:40-47.
64. C roce K, L ibby P. Intertw in ing of thrombosis and inf lamm at ion in atherosclerosis. Curr Op inH em ato,l 2007,14:55-61.
65. G ross GJ, Aucham pach JA. R eperfusion in ju ry:does it exist? JM ol Cell Cardiol 2007,42:12-18.
66. ZhouH, L iu J, Pan P, et a.l Carbon monoxid e inhalation decreased lung injury v ia an t-i in flamm atory and ant-i apop tot iceffects in b rain d eath rats. E xp B iol M ed (M ayw ood),2010,235:1236-1243.
67. A ggarw alA, S chneider DJ, Terrien EF, et a.l Increased coronary arterial release of interleuk in-1 recep tor antagon is t and solub leCD40 ligand ind icat ive of inf lamm at ion associated w ith culprit coronary p laqu es. Am J C ard io,l 2004,93: 6-9
68. Yellon DM, H au sen loy DJ. M yocard ial rep erfu sion in jury. NEngl JM ed, 2007,357:1121-1135.
69. N iccoli G, G iub ilato S, Ru sso E, et a.l P lasm a levels of thromboxan e A2 on admission are associated w ith no-ref low after primary percu taneou s coronary in tervention. Eu rH eart J,2008,29:1843-1850.
70. E itel I, NowakM, S teh l C, et a.l E ndothelin-1 release in acute myocardial infarction as a predictor of long-term prognosis and no reflow assessed by contrast-enhanced magnet icresonan ceimaging. Am H eart J,2010,159: 882-890.
71. N iccoli G, L anza GA, Shaw S, et a.l Endoth elin-1 and acute myocardial infarction:a no-ref low m ed iator after successful percu tan eous m yocardial revascu larizat ion. E ur H eart J,2006,27:1793-1798.
72. TopsakalR, K aya MG, K arakaya E, et a.l Relationsh ip between no-reflow phenomenon and seroton in levels in patients w ith acute ST-elevat ion myocardial in farct ion who underw ent primary percu taneous intervent ion. Anadolu Kard iyo 1 D erg,2010,10:253-259.
73. Sd znerM, Rud iger HA, Sdzn er N1T ran sgen ic m ice overexpressing human are res is tan t to hepatic ischem ia and reperfus ion [J] 1 JH ep ato,l 2002,36(2): 218-2251
74. Sh irito K, Otan i H, Yam am oto F, et al1MK2-/-gene knockout m ouse hearts carry an t-i apoptotic sign al and are resistant to ischem ia reperfus ion inju ry[J] 1 JMo lC ellC ardiao,l 2005,38(1):93-971124-125
75. Ceconi C, Boraso A, Ca rgnoni A, et a.l Ox idativ e stress in cardiovascular d isease:M y th or fact[J]? A rch Biochem Biophys,2003,420:217-221.
76.林燕,黄维义.氧化应激致动脉粥样硬化作用的研究进展[J].黑龙江医学,2004,28(7)：517-519.
77. H eitzer T, Sch linzig T, K rohn K, et a.1 Endo thelia 1 dysfunction, ox idativ e stress,and r isk o f cardiov ascula r events in patients w ith co ronary artery disease [J].C ircula tion,2001,104:2673-2678.
78. M o lav i B, M ehta JL. Ox idativ e stress in cardiovascu lar disease:M o lecular basisof its de leter ious effects, its detect ion, and therapeutic considerat ions [J]. CurrOp in Cardio,1 2004,19(5):488-493.
79. Gr iendling KK, Sorescu D, U shio-FukaiM. NAD (P)H ox idase:Role in cardiovascular bio logy and disease[J]. Circ Res,2000,86:494-501.
80. A zum iH, InoueN, Ohash iY, e t a.1 Superox ide genera tion in directional coronaryatherec tomy specim ens o f patients w ith ang ina pector is:Impor tant ro le o fNAD(P)H ox idase[J]. A r ter iosclerT hromb Vasc Bio,1 2002,22: 1838-1844.
81. Channon KM. Ox idativ e stress and corona ry plaque stab ility [J]. A r ter ioscler Thromb V asc B io,l 2002,22:1751-1752.
82. R a jag opa Ian S,M engXP, R ama samy S, e t a.1R eactive oxyg en spec ies produced by macrophage-der ived foam cells regulate the activ ity o f vascular matrix meta-llo-pro te inases in vitro. Imp lications for atherosclerotic plaque stability [J]. J Clin Inves,t 1996,98:2572-2579.
83.Ndrepepa G, Braun S, Mehilli J, etal.N-terminal pro-brain natriuretic peptide on admission in patients with acute myocardial infarction and correlation with scintigraphic infarct size, efficacy of reperfusion, and prognosis.Am J Cardiol. 2006 Apr 15;97(8):1151-6. Epub 2006 Mar 3.
84.Jarai R, Huber K, Bogaerts K, etal.Plasma N-terminal fragment of the prohormone B-type natriuretic peptide concentrations in relation to time to treatment and Thrombolysis in Myocardial Infarction (TIMI) flow:a substudy of the Assessment of the Safety and Efficacy of a New Treatment Strategy with Percutaneous Coronary Intervention (ASSENT IV-PCI) trial.Am Heart J.2010 Jan;159(1):131-40
85.Hong SN, Ahn Y, Hwang SH, etal.Usefulness of preprocedural N-terminal pro-brain natriuretic peptide in predicting angiographic no-reflow phenomenon during stent implantation in patients with ST-segment elevation acute myocardial infarction. Am J Cardiol.2007 Aug 15;100(4):631-4. Epub 2007 Jun 28.
86.Kim MK, Chung WY, Cho YS, etal.Serum N-terminal pro-B-type natriuretic peptide levels at the time of hospital admission predict of microvascular obstructions after primary percutaneous coronary intervention for acute ST-segment elevation myocardial infarction. J Interv Cardiol. 2011 Feb;24(1):34-41.
87.Pan W, Wang LF, Yu JH, etal .Intracoronary nitroprusside in the prevention of the no-reflow phenomenon in acute myocardial infarction.Chin Med J (Engl). 2009 Nov20;122(22):2718-23.
88 .Demircan S, Yazici M, Hamiseyev C, etal .The role of fibrinolytic system in no-reflow after stenting with and without predilation in patients with acute coronary syndromes: fibrinolysis and no-reflow after coronary stenting. J Thromb Thrombolysis. 2007 Oct;24(2):109-14. Epub 2007 Feb 10.
89.Bonauer A, Carmona G, Iwasaki M,et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice[J]. Science 2009,324:1710-3.
90.Jaffe R, Charron T, Puley G, et al. Microvascular obstruction and the no-reflow phenomenon after percutaneous coronary intervention. Circulation 2008; 117:3152- 6.
91.Northcott PA, Fernandez-L A, Hagan JP, et al. The miR-17/92 polyciSTron is up-regulated in sonic hedgehog-driven medulloblaSTomas and induced by N-myc in sonic hedgehog-treated cerebellar neural precursors[J]. Cancer Res, 2009,69(8):3249.
92.Ye Y, Perez-Polo JR, Qian J, et al. The role of microRNA in modulating myocardial ischemia-reperfusion injury[J]. Physiol Genomics. 2011 May l;43(10):534-42.
93 .Brock M, Trenkmann M, Gay RE, et al. Interleukin-6 modulates the expression of the bone morphogenic protein receptor type II through a novel STAT3-microRNA cluSTer 17/92 pathway[J]. Circ Res, 2009,104(10):1184.
94.Taurino C, Miller WH, McBride MW, et al. Gene expression profiling in whole blood of patients with coronary artery disease [J]. Clin Sci (Lond), 2010,119(8):335-343
95.Iaconetti C, Polimeni A, Sorrentino S. et al. Inhibition of miR-92a increases endothelial proliferation and migration in vitro as well as reduces neointimal proliferation in vivo after vascular injury[J]. Basic Res Cardiol.2012 Sep;107(5):296.
96.Harris TA, Yamakuchi M, Ferlito M, et al. MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1.Proc Natl Acad Sci U S A.2008 Feb 5;105(5):1516-21
97.Fish JE, Santoro MM, Morton SU, et al. miR-126 regulates angiogenic signaling and vascular integrity.Dev Cell.2008 Aug;15(2):272-84.
98.Zhu X, Liu B, Zhou S, et al. Ischemic preconditioning prevents in vivo hyperoxygenation in postischemic myocardium with preservation of mitochondrial oxygen consumption [J]. Am J Physiol Heart Circ Physiol, 2007,923(3):H1442-1450.
99.李袁静,蔡明,陈知水等。建立小鼠急性心肌缺血再灌注损伤模型手术技巧及TTC染色方法讨论[J]。山东医药,2010,50(40)：42-44.
100.Ren A, Yan X, Lu H, et al. Antagonism of endothelin-1 inhibits hypoxia-induced apoptosis in cardiomyocytes [J]. CanJ Physiol Pharmacol, 2008,86:536-540.
101.Xu C, Lu Y, Pan Z, et al. The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis bytargeting HSP60, HSP70 and caspase-9 in cardiomyocytes [J]. J Cell Sci,2007,120 (Pt17):3045-3052.
102.Penna C, Alloatti G, Gallo MP et al Catestatin improves post-ischemic left ventricular function and decreases ischemia/reperfusion injury in heart.Cell Mol Neurobiol.2010 Nov;30(8):1171-9.
103.Liu YH, Zhang M, Liu LM et al.Effects and potential mechanisms of short-term use of simvastatin on myocardial no-reflow after ischemia-reperfusion in rats. Zhong hua Xin Xue Guan Bing Za Zhi.2008 Aug;36(8):729-34.
104.冯国清.黄芪对大鼠心肌缺血再灌注损伤的保护作用[J].中药药理与临床,1997,13(3)：27-29.
105.刘晓春,刘正湘,施静.人参对缺血再灌注大鼠心肌细胞凋亡影响的实验研究[J].中国组织化学与细胞化学杂志,2000,9(3)：261-263.
1.Kiriakidou M, Tan GS, Lamprinaki S, De Planell-Saguer M, Nelson PT & Mourelatos Z (2007) An mRNAm 7G cap binding-like motif within human Ago2 represses translation. Cell 129,1141-1151.
2.Bagga S, Bracht J, Hunter S, Massirer K, Holtz J,Eachus R & Pasquinelli AE (2005) Regulation by let-7and lin-4 miRNAs results in target mRNA degradation.Cell 122,553-563.
3.Humphreys DT, Westman BJ, Martin DI & Preiss T (2005) MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc Natl Acad Sci USA 102,16961-16966.
4.Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993;75:843-854.
5.Berezikov E, Guryev V, van de Belt J, Wienholds E,Plasterk RH & Cuppen E (2005) Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120,21-24.
6. Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, David M. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature. 2007;449:919-922.
7. Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol.2007;5:15-20
8. Tay YM, Tam WL, Ang YS, Gaughwin PM, Yang H, Wang W, Liu R, George J, Ng HH, Perera RJ, Lufkin T, Rigoutsos I, Thomson AM, Lim B. MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1. Stem Cells.2008;26:17-29.
9.Yu LP, Shi LY, Zhang MM, etal,MicroRNA expression profile and pathogenetic initial study in essential hypertension.Zhonghua Xin Xue Guan Bing Za Zhi.2011 Jun;39(6):488-93. Chinese.
10. Castoldi G, Di Gioia CR, Bombardi C,etal.MiR-133a regulates collagen 1A1: potential role of miR-133a in myocardial fibrosis in angiotensin Ⅱ-dependent hypertension. J Cell Physiol.2012 Feb;227(2):850-6. doi:10.1002/jcp.22939
11. Wu WH, Hu CP, Chen XP, etal.MicroRNA-130a mediates proliferation of vascular smooth muscle cells in hypertension. Am J Hypertens.2011 Oct;24(10):1087-93.
12. Zhao Y, R,JF, Li A, etal. Dysreguhtion of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-I-2[J]. Cell,2007.129(2):303-317.
13. Yang B, Lin H。Xiao J。et01. The muscle, specific microRNA miR-I regulates cardiac arrhythmogenie potential by targeting GJAI and KCNJ2[J]. Nat Med, 2007,13(4):486-491.
14. Terentyev D, Belevyeh AE, Terentyeva R, etal. miR-1 overexpres-sion enhances Ca2+release and promotes cardiac arrhythmogenesis by targeting PP2A regulatory subunit B56alpha and causing CaMKII-dependent hyperphesphorylation of RyR2[J]. Cire Res,2009.104(4):514-521.
15. Xiao J,luo X,Lin H. et al. MicroRNA miR-133 represses HERG K+channel expression contributing to QT prolongation in diabetic hearts[J]. J Biol Chem, 2007,282(17):12363-12367.
16. Ono K, Matsumori A, Shioi T, Furukawa Y & Sasayama S (1998) Cytokine gene expression after myocardial infarction in rat hearts:possible implication in left ventricular remodeling. Circulation 98,149-156.
17. Ren XP, Wu J, Wang X, Sartor MA, Qian J, Jones K,Nicolaou P, Pritchard TJ & Fan GC (2009) MicroRNA-320 is involved in the regulation of cardiac ischemia/ reperfusion injury by targeting heat-shock protein 20. Circulation 119, 2357-2366.
18. Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES & Zhang C (2009) MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem 284,29514-29525.
19. Xu C, Lu Y, Pan Z, Chu W, Luo X, Lin H, Xiao J,Shan H, Wang Z & Yang B (2007) The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes. J Cell Sci 120,3045-3052.
20. Shan ZX, Lin QX, Fu YH, Deng CY, Zhou ZL, Zhu JN, Liu XY, Zhang YY, Li Y, Lin SG et al. (2009) Upregulated expression of miR-1/miR-206 in a rat model of myocardial infarction. Biochem Biophys Res Commun 381,597-601.
21. Rabinowits G, Gercel-Taylor C, Day JM, Taylor DD & Kloecker GH (2009) Exosomal microRNA:a diagnostic marker for lung cancer. Clin Lung Cancer 10,42-46.
22. Camussi G, Deregibus MC, Bruno S, Cantaluppi V & Biancone L (2010) Exosomes/micro vesicles as a mechanism of cell-to-cell communication. Kidney Int 78,838-848.
2327.Ji X, Takahashi R, Hiura Y, Hirokawa G, Fukushima Y & Iwai N (2009) Plasma miR-208 as a biomarker of myocardial injury. Clin Chem 55,1944-1949.
24. Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, Qin YW & Jing Q (2010) Circulating microRNA:a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 31,659-666.
25. D'Alessandra Y, Devanna P, Limana F, Straino S, Di Carlo A, Brambilla PG, Rubino M, Carena MC, Spazzafumo L, De Simone M et al. (2010) Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J 31,2765-2773.
26. Cheng Y, Tan N, Yang J, Liu X, Cao X, He P, Dong X, Qin S & Zhang C (2010) A translational study of circulating cell-free microRNA-1 in acute myocardial infarction. Clin Sci (Lond) 119,87-95.
27. Ai J, Zhang R, Li Y, Pu J, Lu Y, Jiao J, Li K, Yu B, Li Z, Wang R et al. (2010) Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biochem Biophys Res Commun 391,73-77.
28. McKinsey TA, Olson EN. Toward transcriptional therapies for the failing heart: chemical screens to modulate genes. J Clin Invest 2005; 115:538-46.
29. van Rooij E, Sutherland LB, Hu N, et 01. A signature pattern Of "stress-responsive micmRNAs that can evoke cardiac hypertrophy and heart failure[J]. Proc Nail Aead Sci USA,2006,103(48):18255.18260.
30. Care A, Catalucci D, Felieetti F, et al. MicrORNA-133 controls cardiac hypertrophy[J]. Nat Med,2007,13(5):613-618.
31. Cheng Y, Ji R, Yue J,a a/. MieroRNAsmaberrantly expressed in hypertrophic heart:do they play a role in cardiac hypertrophy?[J]. Am J Pathol,2007,170(6): 1831-1840.
32. Tatsuguchi M, Seok HY, Callis TE, et al. Expression of mieroRNAs is dynamically regulated during cardiomyocyte hypertrophy[J]. J Mol Cell Cardiol.2007,42(6):1137-1141.
33. Matkovich SJ, Van Booven DJ, Youker KA, Torre-Amione G, Diwan A,Eschenbacher WH, Dorn LE, Watson MA, Margulies KB, Dorn GW. Reciprocal regulation of myocardial microRNAs and messenger RNA in human cardiomyopathy and reversal of the microRNA signature by biomechanical support. Circulation 2009; 119:1263-1271.
34. Schipper ME, van Kuik J, de Jonge N, Dull ens HF, de Weger RA. Changes in regulatory microRNA expression in myocardium of heart failure patients on left ventricular assist device support. J Heart Lung Transplant 2008;27:1282-1285
2. Yellon DM, Hausenloy DJ. Myocardial reperfusion injury [J] NEngl J Med,2007,357(11):1121-1135.
3. ENGLER R L, SCHIMD SCHONBEIN G W, PAVALEC R S.Leu kocyt e capill ary plu gging in m yocar dial isch emia andreperfusion in the dog[J]. Am J Pathol,1983,3:982-987.
4.严勤,苏肇伉,丁文祥.氧自由基清除剂对缺血犬心肌再灌注损伤的保护作用[J].中华胸心血管外科杂志,1995,11(1)：42.
5. ALICIA J K, ROGE R F C, ANIBAL E V. Mit ochond rial p ermeability transit ion and ox idat ive t res s. FEBS let ters [J]. Circ Res,2001,495:12-15.
6. GrossG J, Kersten JR,W arltier DC. Mechan ism s of postisch em ic contractile dysfunct ion[J]. Ann Thorac Su rg,1999,68(5):1898-1904.
7. LOPASCHUK G, ALBERT A E. Regulat ion of carbohydr at emet abolism in i sch emia and reperfusion [J]. Am Heart J,2000,139:S115-S119.
8. HAYASHI H.Pathogen esis and the role of Ca2+ overl oad during myocardial isch emia/reperfus ion [J]. Nagoya J Med Sci,2000,63(3-4):91-98.
9. TE RRY L, VANDEN HOEK, LANC E B, et al. React ive oxygen species released f rom m itochondria du ring b rief hypoxia indu ceprecondit ioning in cardiomyocyt es [J]. J Biol Ch em,1998,273(29):18092-18098.
10. CHUANYU L I, ROBERT M, JAC KSON. React ive s pecies mechanism s of cel lul ar h ypoxia-r eoxygenat ion injury [J]. Am J Physiol Cell Phys iol,2002, 282(2):C227-C241.
11.杨彦玲,师养荣.心肌缺血再灌注损伤研究进展[J].心血管病学进展,2003,24(2)：116-121.
12.梁晚益,黄跃生.线粒体在缺血再灌注细胞损伤中的作用[J].国外医学：生理、病理科学与临床分册,2000,20(5)：364-367.
13. Gotdhab er JI, Q ayyum MSI Oxygen free rad icals and excitat ion con traction coupl ing[J] 1 Ant iox id Redox S igna,l 2000,2(1):55-641
14.吴傅轩,丁文惠,李大元,等1炎症递质在兔心肌缺血再灌注损伤中的作用[J]临床心血管病杂志,2000,16(2)：85-871
15. H am isonWR, Mertis CF, Leathers CR1 Ev idence of collid ividom ycos is in the skeleton of an ancient Ind ia[J]1C irc Res,1997,68:1446-14571
16.丁钢,沈景霞1心肌缺血再灌注损伤中一氧化氮与中性粒细胞之间的关系[J]心血管病学进展,2000,21(5)：290-2921
17.Kiriakidou M, Tan GS, Lamprinaki S, et al. An mRNAm 7G cap binding-like motif within human Ago2 represses translation[J]. Cell.2007,129,1141-1151.
18.Bagga S, Bracht J, Hunter S, et al. Regulation by let-7and lin-4 miRNAs results in target mRNA degradation.Cell.2005,122,553-563.
19.Humphreys DT, Westman BJ, Martin DI et al. MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc Natl Acad Sci USA.2005,102,16961-16966.
20.Lee RC, Feinbaum RL, Ambros V. et al. Elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993;75:843-854.
21.Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell.2005,120,21-24.
22. Pedersen IM, Cheng G, Wieland S, et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature.2007;449:919-922.
23. Kloosterman WP, Lagendijk AK, Ketting RF, et al. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol.2007;5:15-20
24. Tay YM, Tam WL, Ang YS, et al. MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1. Stem Cells.2008;26:17-29.
25.McKinsey TA, Olson EN, et al. Toward transcriptional therapies for the failing heart:chemical screens to modulate genes. J Clin Invest 2005; 115:538-46.
26.van Rooij E, Sutherland LB, Hu N, et al.A signature pattern Of "stress-responsive micmRNAs that can evoke cardiac hypertrophy and heart failure[J]. Proc Nail Aead Sci USA,2006,103(48):18255-18260.
27.Care A, Catalucci D, Felieetti F, et al. MicrORNA-133 controls cardiac hypertrophy[J]. Nat Med,2007,13(5):613-618.
28.Cheng Y, Ji R, Yue J, et al.MieroRNAsmaberrantly expressed in hypertrophic heart:do they play a role in cardiac hypertrophy?[J]. Am J Pathol,2007,170(6): 1831-1840.
29.Tatsuguchi M, Seok HY, Callis TE, et al. Expression of mieroRNAs is dynamically regulated during cardiomyocyte hypertrophy[J]. J Mol Cell Cardiol.2007,42(6):1137-1141.
30. Matkovich SJ, Van Booven DJ, Youker KA, et al. Reciprocal regulation of myocardial microRNAs and messenger RNA in human cardiomyopathy and reversal of the microRNA signature by biomechanical support. Circulation 2009; 119:1263-1271.
31.Schipper ME, van Kuik J, de Jonge N, et al.Changes in regulatory microRNA expression in myocardium of heart failure patients on left ventricular assist device support. J Heart Lung Transplant 2008;27:1282-1285
32.ZhaoY, R,JF, Li A, etal. Dysreguhtion of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-I-2[J]. Cell,2007。129(2):303-317.
33.Yang B, Lin H,Xiao J, et al.The muscle, specific microRNA miR-Ⅰ regulates cardiac arrhythmogenie potential by targeting GJ AI and KCNJ2[J]. Nat Med, 2007,13(4):486-491.
34.Terentyev D, Belevyeh AE, Terentyeva R, etal. MiR-1 overexpres-sion enhances Ca2+release and promotes cardiac arrhythmogenesis by targeting PP2A regulatory subunit B56alpha and causing CaMKII-dependent hyperphesphorylation of RyR2[J]. Cire Res,2009。104(4):514-521.
35.Xiao J,luo X,Lin H. et al. MicroRNA miR-133 represses HERG K+channel expression contributing to QT prolongation in diabetic hearts[J]. J Biol Chem, 2007,282(17):12363-12367.
36.Ono K, Matsumori A, Shioi T, et al. Cytokine gene expression after myocardial infarction in rat hearts:possible implication in left
ventricular remodeling. Circulation.1998,98,149-156.
37.Ren XP, Wu J, Wang X, et al. MicroRNA-320 is involved in the regulation of cardiac ischemia/ reperfusion injury by targeting heat-shock protein 20. Circulation.2009,119,2357-2366.
38.Dong S, Cheng Y, Yang J, et al. MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem.2009,284,29514-29525.
39.Xu C, Lu Y, Pan Z, et al. The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes. J Cell Sci.2007,120,3045-3052.
40.Shan ZX, Lin QX, Fu YH, et al. Upregulated expression of miR-1/miR-206 in a rat model of myocardial infarction. Biochem Biophys Res Commun.2009,381, 597-601.
41.Rabinowits G, Gercel-Taylor C, Day JM et al. Exosomal microRNA:a diagnostic marker for lung cancer. Clin Lung Cancer.2009,10,42-46.
42.Camussi G, Deregibus MC, Bruno S, et al. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int.2010,78,838-848.
43 Ji X, Takahashi R, Hiura Y, et al. Plasma miR-208 as a biomarker of myocardial injury. Clin Chem.2009,55,1944-1949.
44.Wang GK, Zhu JQ, Zhang JT, et al. Circulating microRNA:a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J.2010,31,659-666.
45. D'Alessandra Y, Devanna P, et al. Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J.2010,31,2765-2773.
46.Cheng Y, Tan N, Yang J, et al. A translational study of circulating cell-free microRNA-1 in acute myocardial infarction. Clin Sci (Lond).2010,119,87-95.
47.Ai J, Zhang R, Li Y, et al. Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biochem Biophys Res Commun.2010,391, 73-77.
48.Yu LP, Shi LY, Zhang MM, etal,MicroRNA expression profile and pathogenetic initial study in essential hypertension.Zhonghua Xin Xue Guan Bing Za Zhi.2011 Jun;39(6):488-93. Chinese.
49.Castoldi G, Di Gioia CR, Bombardi C,etal.MiR-133a regulates collagen 1A1: potential role of miR-133a in myocardial fibrosis in angiotensin II-dependent hypertension. J Cell Physiol.2012 Feb;227(2):850-6. doi:10.1002/jcp.22939
50.Wu WH, Hu CP, Chen XP, etal.MicroRNA-130a mediates proliferation of vascular smooth muscle cells in hypertension.Am J Hypertens.2011 Oct;24(10):1087-93.
51.Wang S, Aurora AB, Johnson BA, et al. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell 2008; 15:261-271.
52.van Solingen C, Seghers L, Bijkerk R, et al. ntagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis. J Cell Mol Med 2009;13:1577-1585.
53.Sun X, Zhang M, Sanagawa A, et al. Circulating microRNA-126 in patients with coronary artery disease:correlation with LDL cholesterol.Thromb J.2012 Aug 28;10(1):16.
54.Guangwen Long,* Feng Wang, Quanlu Duan, et al.Human Circulating MicroRNA-1 and MicroRNA-126 as Potential Novel Indicators for Acute Myocardial Infarction Int J Biol Sci.2012; 8(6):811-818. Published online 2012 June 7.
55.Bonauer A, Carmona G, Iwasaki M,et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice[J]. Science 2009,324:1710-3.
56. Wang H, Lu HM, Yang WH et al. Zhong guo Wei Zhong Bing Ji Jiu Yi Xue. The influence of statin therapy on circulating microRNA-92a expression in patients with coronary heart disease [J].2012 Apr,24(4):215-8.
57. Salloum FN, Yin C, Kukreja RC, et al. Role of microRNAs in cardiac preconditioning. J Cardiovasc Pharmacol[J].2010 Dec,56(6):581-8.
58.McKinsey TA, Olson EN. Toward transcriptional therapies for the failing heart: chemical screens to modulate genes[J]. J Clin Invest,2005,115(3):538-546.
59.Steffens S, Montecucco F, Mach F. The inflammatory response as a target to reduce myocardial ischaemia and reperfusion injury[J].Thromb Haemost, 2009,102(2):240-247.
60Ndrepepa G, Braun S, Mehilli J, etal.N-terminal pro-brain natriuretic peptide on admission in patients with acute myocardial infarction and correlation with scintigraphic infarct size, efficacy of reperfusion, and prognosis.Am J Cardiol. 2006 Apr 15;97(8):1151-6. Epub 2006 Mar 3.
61.杨跃进.经皮冠状动脉介入治疗中无再流的诊断、治疗和预防[J].中华心血管病杂志,2004,32(7)：662-664.
62. C arrabba N, Parod i G, Valent i R, et a.l S ign ifican ce of add itional ST segment elevation in patients w ith no reflow afterang iop lasty for acu te myocardial in farct ion. J Am SocE chocardiogr,2007,20:262-269.
63. van d erW alAC, Li X, de Boer O J. T issue factor exp ression in the m orphologic spectrum of vu lnerab le atherosclerotic p laques.Sem in Throm bH emost,2006, 32:40-47.
64. C roce K, L ibby P. Intertw in ing of thrombosis and inf lamm at ion in atherosclerosis. Curr Op inH em ato,l 2007,14:55-61.
65. G ross GJ, Aucham pach JA. R eperfusion in ju ry:does it exist? JM ol Cell Cardiol 2007,42:12-18.
66. ZhouH, L iu J, Pan P, et a.l Carbon monoxid e inhalation decreased lung injury v ia an t-i in flamm atory and ant-i apop tot iceffects in b rain d eath rats. E xp B iol M ed (M ayw ood),2010,235:1236-1243.
67. A ggarw alA, S chneider DJ, Terrien EF, et a.l Increased coronary arterial release of interleuk in-1 recep tor antagon is t and solub leCD40 ligand ind icat ive of inf lamm at ion associated w ith culprit coronary p laqu es. Am J C ard io,l 2004,93: 6-9
68. Yellon DM, H au sen loy DJ. M yocard ial rep erfu sion in jury. NEngl JM ed, 2007,357:1121-1135.
69. N iccoli G, G iub ilato S, Ru sso E, et a.l P lasm a levels of thromboxan e A2 on admission are associated w ith no-ref low after primary percu taneou s coronary in tervention. Eu rH eart J,2008,29:1843-1850.
70. E itel I, NowakM, S teh l C, et a.l E ndothelin-1 release in acute myocardial infarction as a predictor of long-term prognosis and no reflow assessed by contrast-enhanced magnet icresonan ceimaging. Am H eart J,2010,159: 882-890.
71. N iccoli G, L anza GA, Shaw S, et a.l Endoth elin-1 and acute myocardial infarction:a no-ref low m ed iator after successful percu tan eous m yocardial revascu larizat ion. E ur H eart J,2006,27:1793-1798.
72. TopsakalR, K aya MG, K arakaya E, et a.l Relationsh ip between no-reflow phenomenon and seroton in levels in patients w ith acute ST-elevat ion myocardial in farct ion who underw ent primary percu taneous intervent ion. Anadolu Kard iyo 1 D erg,2010,10:253-259.
73. Sd znerM, Rud iger HA, Sdzn er N1T ran sgen ic m ice overexpressing human are res is tan t to hepatic ischem ia and reperfus ion [J] 1 JH ep ato,l 2002,36(2): 218-2251
74. Sh irito K, Otan i H, Yam am oto F, et al1MK2-/-gene knockout m ouse hearts carry an t-i apoptotic sign al and are resistant to ischem ia reperfus ion inju ry[J] 1 JMo lC ellC ardiao,l 2005,38(1):93-971124-125
75. Ceconi C, Boraso A, Ca rgnoni A, et a.l Ox idativ e stress in cardiovascular d isease:M y th or fact[J]? A rch Biochem Biophys,2003,420:217-221.
76.林燕,黄维义.氧化应激致动脉粥样硬化作用的研究进展[J].黑龙江医学,2004,28(7)：517-519.
77. H eitzer T, Sch linzig T, K rohn K, et a.1 Endo thelia 1 dysfunction, ox idativ e stress,and r isk o f cardiov ascula r events in patients w ith co ronary artery disease [J].C ircula tion,2001,104:2673-2678.
78. M o lav i B, M ehta JL. Ox idativ e stress in cardiovascu lar disease:M o lecular basisof its de leter ious effects, its detect ion, and therapeutic considerat ions [J]. CurrOp in Cardio,1 2004,19(5):488-493.
79. Gr iendling KK, Sorescu D, U shio-FukaiM. NAD (P)H ox idase:Role in cardiovascular bio logy and disease[J]. Circ Res,2000,86:494-501.
80. A zum iH, InoueN, Ohash iY, e t a.1 Superox ide genera tion in directional coronaryatherec tomy specim ens o f patients w ith ang ina pector is:Impor tant ro le o fNAD(P)H ox idase[J]. A r ter iosclerT hromb Vasc Bio,1 2002,22: 1838-1844.
81. Channon KM. Ox idativ e stress and corona ry plaque stab ility [J]. A r ter ioscler Thromb V asc B io,l 2002,22:1751-1752.
82. R a jag opa Ian S,M engXP, R ama samy S, e t a.1R eactive oxyg en spec ies produced by macrophage-der ived foam cells regulate the activ ity o f vascular matrix meta-llo-pro te inases in vitro. Imp lications for atherosclerotic plaque stability [J]. J Clin Inves,t 1996,98:2572-2579.
83.Ndrepepa G, Braun S, Mehilli J, etal.N-terminal pro-brain natriuretic peptide on admission in patients with acute myocardial infarction and correlation with scintigraphic infarct size, efficacy of reperfusion, and prognosis.Am J Cardiol. 2006 Apr 15;97(8):1151-6. Epub 2006 Mar 3.
84.Jarai R, Huber K, Bogaerts K, etal.Plasma N-terminal fragment of the prohormone B-type natriuretic peptide concentrations in relation to time to treatment and Thrombolysis in Myocardial Infarction (TIMI) flow:a substudy of the Assessment of the Safety and Efficacy of a New Treatment Strategy with Percutaneous Coronary Intervention (ASSENT IV-PCI) trial.Am Heart J.2010 Jan;159(1):131-40
85.Hong SN, Ahn Y, Hwang SH, etal.Usefulness of preprocedural N-terminal pro-brain natriuretic peptide in predicting angiographic no-reflow phenomenon during stent implantation in patients with ST-segment elevation acute myocardial infarction. Am J Cardiol.2007 Aug 15;100(4):631-4. Epub 2007 Jun 28.
86.Kim MK, Chung WY, Cho YS, etal.Serum N-terminal pro-B-type natriuretic peptide levels at the time of hospital admission predict of microvascular obstructions after primary percutaneous coronary intervention for acute ST-segment elevation myocardial infarction. J Interv Cardiol. 2011 Feb;24(1):34-41.
87.Pan W, Wang LF, Yu JH, etal .Intracoronary nitroprusside in the prevention of the no-reflow phenomenon in acute myocardial infarction.Chin Med J (Engl). 2009 Nov20;122(22):2718-23.
88 .Demircan S, Yazici M, Hamiseyev C, etal .The role of fibrinolytic system in no-reflow after stenting with and without predilation in patients with acute coronary syndromes: fibrinolysis and no-reflow after coronary stenting. J Thromb Thrombolysis. 2007 Oct;24(2):109-14. Epub 2007 Feb 10.
89.Bonauer A, Carmona G, Iwasaki M,et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice[J]. Science 2009,324:1710-3.
90.Jaffe R, Charron T, Puley G, et al. Microvascular obstruction and the no-reflow phenomenon after percutaneous coronary intervention. Circulation 2008; 117:3152- 6.
91.Northcott PA, Fernandez-L A, Hagan JP, et al. The miR-17/92 polyciSTron is up-regulated in sonic hedgehog-driven medulloblaSTomas and induced by N-myc in sonic hedgehog-treated cerebellar neural precursors[J]. Cancer Res, 2009,69(8):3249.
92.Ye Y, Perez-Polo JR, Qian J, et al. The role of microRNA in modulating myocardial ischemia-reperfusion injury[J]. Physiol Genomics. 2011 May l;43(10):534-42.
93 .Brock M, Trenkmann M, Gay RE, et al. Interleukin-6 modulates the expression of the bone morphogenic protein receptor type II through a novel STAT3-microRNA cluSTer 17/92 pathway[J]. Circ Res, 2009,104(10):1184.
94.Taurino C, Miller WH, McBride MW, et al. Gene expression profiling in whole blood of patients with coronary artery disease [J]. Clin Sci (Lond), 2010,119(8):335-343
95.Iaconetti C, Polimeni A, Sorrentino S. et al. Inhibition of miR-92a increases endothelial proliferation and migration in vitro as well as reduces neointimal proliferation in vivo after vascular injury[J]. Basic Res Cardiol.2012 Sep;107(5):296.
96.Harris TA, Yamakuchi M, Ferlito M, et al. MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1.Proc Natl Acad Sci U S A.2008 Feb 5;105(5):1516-21
97.Fish JE, Santoro MM, Morton SU, et al. miR-126 regulates angiogenic signaling and vascular integrity.Dev Cell.2008 Aug;15(2):272-84.
98.Zhu X, Liu B, Zhou S, et al. Ischemic preconditioning prevents in vivo hyperoxygenation in postischemic myocardium with preservation of mitochondrial oxygen consumption [J]. Am J Physiol Heart Circ Physiol, 2007,923(3):H1442-1450.
99.李袁静,蔡明,陈知水等。建立小鼠急性心肌缺血再灌注损伤模型手术技巧及TTC染色方法讨论[J]。山东医药,2010,50(40)：42-44.
100.Ren A, Yan X, Lu H, et al. Antagonism of endothelin-1 inhibits hypoxia-induced apoptosis in cardiomyocytes [J]. CanJ Physiol Pharmacol, 2008,86:536-540.
101.Xu C, Lu Y, Pan Z, et al. The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis bytargeting HSP60, HSP70 and caspase-9 in cardiomyocytes [J]. J Cell Sci,2007,120 (Pt17):3045-3052.
102.Penna C, Alloatti G, Gallo MP et al Catestatin improves post-ischemic left ventricular function and decreases ischemia/reperfusion injury in heart.Cell Mol Neurobiol.2010 Nov;30(8):1171-9.
103.Liu YH, Zhang M, Liu LM et al.Effects and potential mechanisms of short-term use of simvastatin on myocardial no-reflow after ischemia-reperfusion in rats. Zhong hua Xin Xue Guan Bing Za Zhi.2008 Aug;36(8):729-34.
104.冯国清.黄芪对大鼠心肌缺血再灌注损伤的保护作用[J].中药药理与临床,1997,13(3)：27-29.
105.刘晓春,刘正湘,施静.人参对缺血再灌注大鼠心肌细胞凋亡影响的实验研究[J].中国组织化学与细胞化学杂志,2000,9(3)：261-263.
1.Kiriakidou M, Tan GS, Lamprinaki S, De Planell-Saguer M, Nelson PT & Mourelatos Z (2007) An mRNAm 7G cap binding-like motif within human Ago2 represses translation. Cell 129,1141-1151.
2.Bagga S, Bracht J, Hunter S, Massirer K, Holtz J,Eachus R & Pasquinelli AE (2005) Regulation by let-7and lin-4 miRNAs results in target mRNA degradation.Cell 122,553-563.
3.Humphreys DT, Westman BJ, Martin DI & Preiss T (2005) MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc Natl Acad Sci USA 102,16961-16966.
4.Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993;75:843-854.
5.Berezikov E, Guryev V, van de Belt J, Wienholds E,Plasterk RH & Cuppen E (2005) Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120,21-24.
6. Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, David M. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature. 2007;449:919-922.
7. Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol.2007;5:15-20
8. Tay YM, Tam WL, Ang YS, Gaughwin PM, Yang H, Wang W, Liu R, George J, Ng HH, Perera RJ, Lufkin T, Rigoutsos I, Thomson AM, Lim B. MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1. Stem Cells.2008;26:17-29.
9.Yu LP, Shi LY, Zhang MM, etal,MicroRNA expression profile and pathogenetic initial study in essential hypertension.Zhonghua Xin Xue Guan Bing Za Zhi.2011 Jun;39(6):488-93. Chinese.
10. Castoldi G, Di Gioia CR, Bombardi C,etal.MiR-133a regulates collagen 1A1: potential role of miR-133a in myocardial fibrosis in angiotensin Ⅱ-dependent hypertension. J Cell Physiol.2012 Feb;227(2):850-6. doi:10.1002/jcp.22939
11. Wu WH, Hu CP, Chen XP, etal.MicroRNA-130a mediates proliferation of vascular smooth muscle cells in hypertension. Am J Hypertens.2011 Oct;24(10):1087-93.
12. Zhao Y, R,JF, Li A, etal. Dysreguhtion of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-I-2[J]. Cell,2007.129(2):303-317.
13. Yang B, Lin H。Xiao J。et01. The muscle, specific microRNA miR-I regulates cardiac arrhythmogenie potential by targeting GJAI and KCNJ2[J]. Nat Med, 2007,13(4):486-491.
14. Terentyev D, Belevyeh AE, Terentyeva R, etal. miR-1 overexpres-sion enhances Ca2+release and promotes cardiac arrhythmogenesis by targeting PP2A regulatory subunit B56alpha and causing CaMKII-dependent hyperphesphorylation of RyR2[J]. Cire Res,2009.104(4):514-521.
15. Xiao J,luo X,Lin H. et al. MicroRNA miR-133 represses HERG K+channel expression contributing to QT prolongation in diabetic hearts[J]. J Biol Chem, 2007,282(17):12363-12367.
16. Ono K, Matsumori A, Shioi T, Furukawa Y & Sasayama S (1998) Cytokine gene expression after myocardial infarction in rat hearts:possible implication in left ventricular remodeling. Circulation 98,149-156.
17. Ren XP, Wu J, Wang X, Sartor MA, Qian J, Jones K,Nicolaou P, Pritchard TJ & Fan GC (2009) MicroRNA-320 is involved in the regulation of cardiac ischemia/ reperfusion injury by targeting heat-shock protein 20. Circulation 119, 2357-2366.
18. Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES & Zhang C (2009) MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem 284,29514-29525.
19. Xu C, Lu Y, Pan Z, Chu W, Luo X, Lin H, Xiao J,Shan H, Wang Z & Yang B (2007) The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes. J Cell Sci 120,3045-3052.
20. Shan ZX, Lin QX, Fu YH, Deng CY, Zhou ZL, Zhu JN, Liu XY, Zhang YY, Li Y, Lin SG et al. (2009) Upregulated expression of miR-1/miR-206 in a rat model of myocardial infarction. Biochem Biophys Res Commun 381,597-601.
21. Rabinowits G, Gercel-Taylor C, Day JM, Taylor DD & Kloecker GH (2009) Exosomal microRNA:a diagnostic marker for lung cancer. Clin Lung Cancer 10,42-46.
22. Camussi G, Deregibus MC, Bruno S, Cantaluppi V & Biancone L (2010) Exosomes/micro vesicles as a mechanism of cell-to-cell communication. Kidney Int 78,838-848.
2327.Ji X, Takahashi R, Hiura Y, Hirokawa G, Fukushima Y & Iwai N (2009) Plasma miR-208 as a biomarker of myocardial injury. Clin Chem 55,1944-1949.
24. Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, Qin YW & Jing Q (2010) Circulating microRNA:a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 31,659-666.
25. D'Alessandra Y, Devanna P, Limana F, Straino S, Di Carlo A, Brambilla PG, Rubino M, Carena MC, Spazzafumo L, De Simone M et al. (2010) Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J 31,2765-2773.
26. Cheng Y, Tan N, Yang J, Liu X, Cao X, He P, Dong X, Qin S & Zhang C (2010) A translational study of circulating cell-free microRNA-1 in acute myocardial infarction. Clin Sci (Lond) 119,87-95.
27. Ai J, Zhang R, Li Y, Pu J, Lu Y, Jiao J, Li K, Yu B, Li Z, Wang R et al. (2010) Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biochem Biophys Res Commun 391,73-77.
28. McKinsey TA, Olson EN. Toward transcriptional therapies for the failing heart: chemical screens to modulate genes. J Clin Invest 2005; 115:538-46.
29. van Rooij E, Sutherland LB, Hu N, et 01. A signature pattern Of "stress-responsive micmRNAs that can evoke cardiac hypertrophy and heart failure[J]. Proc Nail Aead Sci USA,2006,103(48):18255.18260.
30. Care A, Catalucci D, Felieetti F, et al. MicrORNA-133 controls cardiac hypertrophy[J]. Nat Med,2007,13(5):613-618.
31. Cheng Y, Ji R, Yue J,a a/. MieroRNAsmaberrantly expressed in hypertrophic heart:do they play a role in cardiac hypertrophy?[J]. Am J Pathol,2007,170(6): 1831-1840.
32. Tatsuguchi M, Seok HY, Callis TE, et al. Expression of mieroRNAs is dynamically regulated during cardiomyocyte hypertrophy[J]. J Mol Cell Cardiol.2007,42(6):1137-1141.
33. Matkovich SJ, Van Booven DJ, Youker KA, Torre-Amione G, Diwan A,Eschenbacher WH, Dorn LE, Watson MA, Margulies KB, Dorn GW. Reciprocal regulation of myocardial microRNAs and messenger RNA in human cardiomyopathy and reversal of the microRNA signature by biomechanical support. Circulation 2009; 119:1263-1271.
34. Schipper ME, van Kuik J, de Jonge N, Dull ens HF, de Weger RA. Changes in regulatory microRNA expression in myocardium of heart failure patients on left ventricular assist device support. J Heart Lung Transplant 2008;27:1282-1285
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |