【作者】 王翠平；

【导师】 严金川；

【作者基本信息】 江苏大学 ， 内科学， 2009， 硕士

【摘要】 急性冠脉综合征(ACS)是由于冠状动脉不稳定动脉粥样硬化(AS)斑块破裂继发血栓形成,导致管腔严重狭窄或闭塞引起急性或亚急性心肌缺血的临床综合征。包括不稳定心绞痛、ST段抬高的心肌梗死和非ST段抬高心肌梗死。众多资料显示:ACS患病率逐年的增高,目前已成为心血管疾病的“头号无声杀手”。随着对ACS发生机制认识的不断深入,对ACS规范化治疗的逐步完善,特别是近几年加强了抗血小板、稳定斑块措施以及早期的介入治疗使该综合征的死亡率明显降低。众多的研究显示,AS斑块局部的免疫介导、炎症反应和血栓形成是导致ACS发生、发展的中心环节,斑块的稳定性是影响ACS发生、发展的主要决定性因素。斑块中各种炎症因子和细胞因子的相互作用加速斑块的形成和破裂,其中CD40-CD40配体相互作用甚是引起人们的重视。它们是一对互补的受体—配体细胞跨膜糖蛋白,属于肿瘤坏死因子受体(TNFR)和TNFR超家族成员,在体液免疫和细胞免疫中传递重要的细胞信号通路。近年来的研究发现,这一对受体-配体轴的表达和相互作用参与了AS斑块的发生、发展并进一步激活血小板,在ACS的发生中起关键作用,逐渐受到国内、外学者关注。CD40-CD40L基因突变或表达异常与某些炎症反应,自身免疫性疾病和免疫缺陷病的发生密切相关。有研究发现CD40基因启动子区域-1位C/T与CD40蛋白的表达有关,与众多免疫炎症性疾病相关。本实验在原有的工作基础上采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测了ACS患者、稳定型心绞痛患者(SA)及冠脉正常者CD40-1C/T基因多态性并测序鉴定,采用流式细胞术检测血单核细胞表达CD40水平,探讨CD40基因启动子区域-1C/T单核苷酸多态性位点(SNP)与急性冠脉综合征(ACS)的易感相关性,并结合血单核细胞表达CD40水平,探讨其机制是否与基因位点的改变引起下游蛋白的改变,从而增加疾病的易患风险有关。结果发现ACS患者CC基因型及C等位基因频率均明显高于SA患者及对照组(32.3%vs 19.5%vs 15.6;58.7%vs 47.2%vs 41.8%),而TT基因型和T等位基因频率明显低于SA和对照组(14.8%vs 25.1%vs 32.0%;41.3%vs 52.8%vs 58.2%)(P＜0.01)。基因型及等位基因频率在SA组与对照组无统计学差异(P=0.198;P=0.092)。C等位基因的携带者ACS的患病风险相对SA增加了1.841倍(OR=1.841,95%CI:1.390-2.437),相对于对照组患病风险增加了1.877倍(OR=1.877,95%CI:1.402-2.515)。各组外周血单核细胞CD40表达的平均荧光强度分别为(24.8±7.6,25.4±9.1,65.6±15.4),其中ACS组明显高于SA组及对照组(P＜0.01),而SA组和对照组无显著差异(P＞0.05)。ACS组中各基因型CD40表达分别为(74.6±8.7,56.8±6.7,46.3±9.8),CC基因型患者单核细胞CD40的表达高于CT型及TT型(P＜0.01),CT基因型高于TT基因型(P＜0.01)。CD40基因启动子区域的-1C/T单核苷酸多态性(SNP)位点,C等位基因明显增加了ACS的患病风险,其机制可能与其增加了CD40蛋白的表达有关。近几年人们认识到,决定ACS的因素不是粥样斑块的数量、分布和大小,而是外在触发因素和斑块内在特性相互作用导致斑块破裂的结局。冠状动脉造影提示约有半数急性血栓病变其狭窄程度仅为轻度或中度,ACS的发生与冠脉粥样硬化斑块的不稳定性有关,而与冠脉的狭窄程度无关。如何早期预测不稳定斑块,早期采取干预手段,减少急性心血管事件的发生成为目前研究的热点。冠脉造影结果显示冠脉粥样硬化呈现复杂狭窄较光滑狭窄可更迅速发展,甚至发生完全闭塞病变,提示斑块不稳定。本实验在揭示CD40-1C/T多态性与ACS关系的基础上,结合冠脉造影结果分析CD40-1C/T基因多态性与冠脉复杂狭窄的关系,进一步探讨其与不稳定斑块的关系。结果发现:复杂狭窄组CC基因型频率明显高于光滑狭窄组及对照组(29.4%vs 15.3%vs 15.6%),同时TT基因型明显低于光滑狭窄组和对照组(16.9%vs 29.0%32.0%)(P＜0.01)。复杂狭窄组的C等位基因频率明显高于光滑狭窄组和对照组(56.2%vs 43.1%vs 41.8%)(P＜0.01)。C等位基因与斑块的形态明显相关,增加了复杂狭窄的发生风险(较光滑狭窄组相比OR=1.69795%CI:1.273-2.261;较对照组相比OR=1.793 95%CI:1.410-2.281)。而基因型及等位基因频率在光滑狭窄组和对照组之间无显著差异(P=0.725;P=0.814)。CD40基因启动子区域的-1C/T单核苷酸多态性(SNP)位点与冠脉斑块的不稳定性相关,C等位基因明显增加了斑块的易损风险。总之,本实验结果表明CD40-1C/T基因多态性与急性冠脉综合征相关,增加了ACS的易感性,其可能机制与其增加了CD40蛋白的表达有关,从而影响体内的炎症反应,增加了动脉粥样硬化斑块的不稳定性,增加了ACS的患病风险。更多还原

【Abstract】 Acute coronary syndrome(ACS) encompasses a spectrum of coronary artery diseases,including unstable angina,ST segment elevation myocardial infarction (STEMI;often referred to as "Q-wave myocardial infarction"),and non-ST segment elevation myocardial infarction(NSTEMI;often referred to as "non-Q-wave myocardial infarction").It results from the rupture of atherosclerotic plaques,leading to coronary thrombosis.ACS is the leading cause of morbidity and mortality of the heart disease in the world.With the researches in the mechanism of ACS and the therapies such as anti-platelet,stabilizing the plaque and the early intervention therapy, the mortality of the syndrome has significantly decreased in this decade.Evidences suggested that inflammatory and immunologic mediators and reactions in atherosclerosis plaque and thrombus formation may play a critical role in the pathogenesis and the development of the ACS.Vulnerable plaque was thought to be more crucial determinants of ACS rather than the extent of stenosis.The interaction of inflammatory and immunologic mediators in atherosclerosis plaque triggered the formation and the development of the atherosclerosis plaque.CD40-CD40L is a pair of typeⅡmembrane glycoprotein with homology to tumor necrosis factor receptor family.They play an important role in humoral and cell-mediated immune responses giving rise to inflammatory responses.Recently,increasing evidences showed that the CD40-CD40L interaction plays a crucial role in the pathogenesis of atherosclerosis and coronary artery disease.Researchers have demonstrated that gene mutations or abnormal expression of CD40-CD40L could lead to immunodeficiency and autoimmune diseases.A C/T in the 5’ untranslated region of CD40 located at the -1 position within the Kozak sequence(rs1883832) has been associated with CD40 protein expression.It has been found to associate with diseases related with inflammatory responses.Based on our previous researches,we included patients with either ACS(n=223) or stable angina(SA,n=251) and controls(n=225) in this study.The gene polymorphism was measured by using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing.The expression of CD40 on monocytes was analyzed by indirect-immonofluorescence flow cytometry.The aim of our study is to investigate whether a CD40 gene(-1C/T) single nucleotide polymorphism(SNP) is associated with ACS and whether the mechanism is associated with the up-regulation of CD40 expression.The frequency of the CC genotype of CD40 gene in the ACS group is significantly higher than in the SA and control groups(32.3%vs 19.5%vs 15.6%,P＜0.01),while the TT genotype frequency in the ACS group is significantly lower than that of the SA and control groups((14.8%vs 25.1%vs 32.0%,P＜0.01).The C allele frequency of the ACS group is higher than that of the SA and control groups(58.7%vs 47.2%vs 41.8%,P＜0.01). The C allele increased the risk of ACS as compared with the SA group(OR=1.841, 95%CI:1.390-2.437) and the control groups(OR=1.877,95%CI:1.402-2.515).No significant differences in genotypes or allele frequencies were found between the SA and control groups(P=0.198;P=0.092).The expression of CD40 on monocytes in ACS was higher compared with SA and controls(24.8±7.6 vs 25.4±9.1 vs 65.6±15.4,P＜0.01).In ACS,patients with C alleles carriers had significant higher CD40 expression than the T allele carriers(74.6±8.7 vs 56.8±6.7 vs 46.3±9.8,P＜0.01). The results suggested that the CD40 gene(-1C/T) polymorphism was associated with ACS in Chinese Han people.The -1C allele gene carriers had a higher expression of CD40 and an increased risk of suffering from ACS.It may be related to the up-regulation expression of CD40.In recent years,the composition and morphometry of the vulnerable plaque are thought to be more crucial determinants of ACS likelihood than the extent of stenosis. Angiographically visible complex lesions are associated with plaque vulnerability and have been shown to correlate strongly with clinical severity of conory artery diseases. Compared with smooth lesions,complex lesions are associated with rapid disease progression and a higher restenosis rate after percutaneous transluminal coronary angioplasty,probably reflecting a tendency toward thrombogenesis or further plaque disruption,or both.In this work,we investigated the relationship between the SNP(-1C/T) and the unstable coronary atherosclerotic plaque according to the morphological division of the plaque.The frequency of the CC genotype of CD40 gene in the complex lesions group is significantly higher than that in the smooth lesions and control groups(29.4% vs 15.3%vs 15.6%),while the TT genotype frequency in the complex lesions group is significantly lower than that of the smooth lesion and control groups(16.9%vs 29.0% 32.0%,P＜0.01).The C allele frequency of the complex group is higher than that of the smooth lesions and control groups(56.2%vs 43.1%vs 41.8%,P＜0.01).The C allele increased the risk of happening of the complex lesions as compared with the smooth lesions(OR=1.697,95%CI:1.273-2.261).No significant differences in genotypes or allele frequencies were found between the smooth lesions and control groups(P=0.725;P=0.814).CD40-1C/T polymorphism was associated with unstable coronary atherosclerotic plaque.The C allele frequency increased the risk of the vulnerable plaque to rupture.In conclusion,our findings indicated that the CD40 gene(-1C/T) polymorphism was associated with ACS in South Chinese Han People.It possibly increases the expression of CD40 and results in the inflammatory and immune responses leading to the vulnerable plaque to rupture.更多还原