【作者】 刘文卫；

【导师】 唐其柱；

【作者基本信息】 武汉大学 ， 内科学， 2010， 博士

【摘要】 背景动脉粥样硬化是炎症相关疾病,与粥样硬化相关的细胞因子和细胞因子网络及其炎症瀑布的发现和功能性研究日益受到重视。白介素18(interleukin 18,IL-18)就是这些与粥样硬化相关的细胞因子之一。IL-18具有固有性和适应性免疫细胞调节自然免疫能力,是一种多效促炎症细胞因子。它最初命名为干扰素y (interferon-gamma, IFN-γ)诱导因子,可刺激T淋巴细胞和自然杀伤细胞IFN-γ产生,这两者与粥样硬化斑块关系密切。IL-18可通过p38MAPK-NF-kappaB-PTEN信号系统和MMP-9依赖方式引起冠状动脉平滑肌细胞迁移,诱导内皮细胞凋亡,在内膜斑块形成中起重要作用,并加速粥样硬化的形成和斑块易损。研究证实,血浆IL-18浓度在急性冠脉综合征患者中明显升高,并且可导致心肌功能受损,介导血管斑块易损和破裂,是冠状动脉事件的独立预后因素。在无症状患者中,IL-18不是粥样硬化的独立危险因素；但是与颈动脉稳定性斑块相比较,IL-18 mRNA在不稳定斑块患者中的表达明显增高。合并2型糖尿病的冠心病患者高死亡率越来越受到人们的关注,研究发现,与不合并糖尿病的冠心病患者相比,合并2型糖尿病的男性和女性冠心病患者死亡率均明显增高,这类患者更易出现疾病再发、血管再狭窄及冠脉血运重建术后恶性心血管事件。有证据表明,炎症反应在动脉粥样硬化和2型糖尿病的发生发展中起着至关重要的作用。作为一种促炎症因子,IL-18可能参与了动脉粥样硬化和2型糖尿病的发生发展。颈动脉粥样硬化(carotid atherosclerosis, CAS)作为全身动脉粥样硬化的一个表现,与冠状动脉粥样硬化有着密切的联系；研究证实,通过测定颈动脉IMT及斑块可间接诊断冠心病及其病变的范围和严重程度。经皮冠状动脉介入治疗(percutaneous coronary intervention therapy, PCI)是目前冠心病患者临床上重要的治疗方法之一,PCI能迅速将急性冠脉综合征患者的梗死相关动脉或病变血管开通,恢复前向性血流,有效地降低死亡率,但由此引起的炎性反应以及不良心血管事件也日益受到广泛的关注。炎性反应对心脏有损伤与保护双重作用,也可能是PCI后再狭窄和发生其他心血管事件的重要机制。近期研究发现IL-18基因变异可影响冠心病(coronary artery disease, CAD)患者血液中IL-18水平和临床结局,与年龄相关的心功能损害密切相关,并影响单核细胞IL-18的表达。已有研究证实IL-18基因启动子区-137G/C (RS187238)位点单核苷酸基因多态性(single nucleotide polymorphisms, SNPs)可影响IL-18和IFN-γ的表达。然而IL-18启动子单核苷酸基因多态性与冠心病的相关性并无定论。目的本研究以中国襄樊地区汉族人群中诊断或疑诊为冠心病,经冠状动脉造影确诊或排除冠心病者作为病例组或对照组。本研究旨在：1.评价血清IL-18、肿瘤坏死因子a (TNFa)和白介素-10(IL-10)水平与急性冠脉综合征的关系及其临床意义；2.评价冠心病合并2型糖尿病患者血清IL-18水平及其临床意义；3.探讨冠心病患者颈动脉内膜-中层厚度(carotid artery intima-media thickness, IMT)及斑块与血清IL-18浓度的临床相关性；4.观察经皮冠状动脉介入治疗(PCI)术前与术后血清白细胞介素18(IL-18)的变化,并分析其临床意义；5.采用LDR-PCR技术,检测IL-18基因启动子区的一个功能性-137 G/C(rs187238)位点单核苷酸基因多态性,并分析其与血浆IL-18浓度的相关性,以及在冠心病中的临床价值。方法1.选取住我院心内科冠心病患者91例,非冠心病患者55例,根据临床表现分为急性冠脉综合征(ACS)组,稳定型心绞痛(SAP)组及对照组。评价冠状动脉狭窄程度至少需2次正投影摄片,以最大的狭窄程度确定粥样硬化的严重程度。狭窄节段直径和参照的正常血管测量后,狭窄严重度计算公式：%狭窄=(狭窄部位管腔直径／参照血管直径)×100%。狭窄<50%为对照组,狭窄在50%或以上为病例组(狭窄50%～69%为病例1组；70%～89%为病例2组；狭窄>90%或闭塞为病例3组)。冠状动脉造影前从穿刺动脉抽血。ELISA法测定血清IL-18、TNFa和IL-10浓度并分析。2.选取冠状动脉造影确诊的冠心病患者127例,根据是否合并2型糖尿病分为伴2型糖尿病组(42例)和不伴糖尿病组(85例),以冠状动脉造影排除冠心病者(55例)作为对照,ELISA法测定血清IL-18浓度。3.选取126例经选择性冠状动脉造影检查的患者,依据狭窄程度分为冠心病组(狭窄50%或以上)及对照组(狭窄<50%),行颈动脉超声检查,测量颈动脉内膜中层厚度并观察粥样斑块发生情况；冠状动脉造影前抽取股动脉血,测血清IL-18水平。4.检测87例冠心病患者经皮冠状动脉介入治疗术前及术后12h,术后2周血清IL-18变化,并随访术后12个月内的心血管事件,比较术前、术后以及发生不良心血管事件组与未发生组之间血清IL-18变化的差异。5.用LDR-PCR技术检测并分析了中国襄樊地区汉族人群中241例冠状动脉造影证实的冠心病患者和145例对照组患者的IL-18基因启动子区-137G／C(RS187238)位点单核苷酸基因多态性,并检测各组IL-18水平。结果1.急性冠脉综合征(ACS)组和稳定性心绞痛(SAP)组血清IL-18和TNFa水平高于对照组,有统计学显著性差异(P<0.01),而血清IL-10低于对照组,有统计学显著性差异(P<0.01)。与稳定性心绞痛(SAP)组相比,急性冠脉综合征(ACS)组血清IL-18和TNFα水平明显升高,有统计学显著性差异(P<0.01),而血清IL-10水平则显著降低有统计学显著性差异(P<0.01)。随着冠脉狭窄程度的加重,IL-18水平逐渐增高,病例2组和病例3组均明显高于病例1组,均有统计学显著性差异(P<0.01),研究3组IL-18水平虽高于研究2组,但无统计学显著性差异(P>0.05)；不同狭窄程度的3组冠心病患者TNFα和IL-10水平无统计学显著性差异(P>0.05)。2.冠心病合并2型糖尿病组患者血清IL-18水平高于不伴糖尿病组者,有统计学显著性差异(369.1±113.25 vs 275.9±85.74,P<0.01)。3.冠心病组颈动脉内膜中层厚度、斑块发生率、血清炎性标记物IL-18均比对照组增高,两组间均有统计学显著性差异(P<0.05)。直线相关分析颈动脉内膜中层厚度与IL-18相关性,发现颈动脉内膜中层厚度与IL-18呈正相关,有统计学显著性差异(r=0.251,t=2.09,P=0.034)。冠心病组中颈动脉斑块多发生在颈动脉分叉部及其附近颈总动脉部位,少数发生在颈总动脉起始部或颈内动脉起始段。超声表现为软斑、硬班、钙化斑或复合性斑块,各组间比较,IL-18水平无统计学显著性差异(P>0.05)。4.PCI组术前、术后12h和术后2周的IL-18水平全部明显高于冠脉造影组,有显著性统计学差异(P<0.01)；PCI组术后12h的IL-18水平明显高于术前水平,有显著性统计学差异(P<0.01)；PCI组术后2周的IL-18水平明显低于术后12h和术前的水平,有显著性统计学差异(P<0.01)；冠脉造影组造影前后IL-18水平无显著性统计学差异(P>0.05)。发生不良心血管事件组7例,其中短阵室速3例、室颤1例、II度-II型房室传导阻滞1例、Ⅲ度房室传导阻滞1例、急性左心衰1例；以上并发症经过相应处理后症状缓解。将该7例患者PCI前、术后12h IL-18的差值与发生不良心血管事件之间做相关分析,证实术前术后12h的IL-18差值与不良心血管事件的发生呈正相关,有统计学显著性意义(r=0.86,t=361,P<0.05)。5.全部研究者中,基因型分布与高血压病(P=0.043)和体重指数(P=0.034)有统计学显著性差异(P<0.05),但是与其他冠心病的危险因素(年龄、性别、糖尿病、血脂异常、吸烟等)比较无统计学显著性差异(P>0.05)。病例组(CAD+)G等位基因频率(频率=0.90)与对照组(频率=0.83)比较有统计学显著性差异(P=0.004)；并且病例组GG基因型频率(频率=0.81)高于与对照组(频率=0.63),两组比较有统计学显著性差异(P=0.005)。多支病变(MVD)患者G等位基因频率或GG基因型频率与单支病变(SVD)比较无统计学显著性差异(P>0.05)。病例组(CAD+)含GG纯合子基因型的患者中,其IL-18蛋白浓度水平高于CG杂合子和CC纯合子合并后患者IL-18蛋白水平,有统计学显著性差异(P=0.000)；对于对照组(CAD-)同样如此。结论IL-18、TNFa和IL-10可以反映动脉粥样硬化斑块的严重程度和稳定性状态,可作为监测病情的临床生化指标。合并2型糖尿病的冠心病患者血清IL-18水平高,可能是其易发生致死性心血管事件的重要原因,对这类人群需积极采取措施进行二级预防。颈动脉内膜中层厚度、斑块发生率、血清IL-18水平的检测可作为定量检测冠状动脉粥样硬化,进而判断冠心病危险度的良好指标。冠状动脉介入治疗术后存在炎性反应,IL-18是反映介入治疗后早期炎性反应的敏感指标,它的变化程度对不良心血管事件的发生可能具有预测价值,介入治疗加剧了局部炎症反应,并随着血管开通炎症反应逐渐减弱。IL-18基因启动子区-137G／C单核苷酸基因多态性可影响血浆中IL-18蛋白水平和冠心病的发生,表明IL-18可能参与动脉粥样硬化及其并发症的发生和发展。总之,IL-18与冠心病关系密切,是冠心病诊治过程中极有前景的靶点。更多还原

【Abstract】 BackgroundAtherosclerosis is clearly an inflammatory disease and does not result simply from the accumulation of lipids. The discovery and functional assessment of cytokines and cytokine networks involved in the inflammatory cascade are receiving growing emphasis.Interleukin 18 (IL-18) is a pleiotropic proinflammatory cytokine that has immunomodulatory effects on both the innate and the adaptive immune systems. Originally identified as an interferon-gamma inducing factor, it stimulates interferon-gamma production in T lymphocytes and natural killer cells, both of which are essential for atherosclerotic plaque progression and stability. IL-18 plays a critical role for in neointimal formation, induces endothelial cell apoptosis via activating p38MAPK-NF-kappaB-PTEN signal transduction pathway and human coronary artery smooth muscle cell migration in matrix metalloproteinase-9-dependent manner, and accelerates atherosclerosis and plaque vulnerability. Plasma IL-18 concentrations are increased in patients with acute coronary syndromes and correlate with the severity of myocardial dysfunction, and plasma IL-18 levels are an independent predictor of coronary events, which suggest that IL-18 mediates later events in plaque unstability and eventual disruption. IL-18 is not an independent risk factors for atherosclerosis in asymptomatic individuals, but IL-18 mRNA is expressed at higher levels in unstable human carotid plaques compared with stable plaques.Among the risk factors for coronary artery disease, diabetes mellitus has a privileged position. Cardiovascular complications are the leading cause of mortality in patients with diabetes mellitus. Diabetes mellitus is a strong independent predictor for both death and myocardial infarction (MI).Increased carotid artery intima-media thickness is a marker of coronary vascular disease which can be measured non-invasively and may be used as a biological marker of coronary artery disease. In addition, an increased carotid artery intima-media thickness may have considerable importance as a screening tool for coronary atheroma.Recently, variations within the IL-18 gene have been shown to influence circulating levels of IL-18 and clinical outcome in subjects with coronary artery disease and play an active role in age-related functional impairment, as well as to affect IL-18 production capability by monocytes. It has been identified single nucleotide polymorphisms (SNPs) in the promoter of IL-18 gene at the position-137G/C (RS187238) can influence the expression of IL-18 and also potentially of IFN-gamma. However, the relationship between the functional promoter polymorphism and coronary artery disease has not been clearly defined.Objectives1 To study the relations of the levels of serum interleukin-18, tumor necrosis factor-a and interleukin 10 with acute coronary syndrome and their clinical significance.2 To investigate the expression and significance of interleukin-18 in coronary heart disease patients with type 2 diabetes mellitus.3 To investigate the clinical relationship of carotid artery intima-media thickness, atherosclerotic plaque and serum interleukin-18 levels in patients with coronary heart diseases.4 To investigate the changes of serum interleukin-18 before and after percutaneous coronary intervention therapy for exploring the Clinical significance.5 To investigate the clinical significance of the single nucleotide polymorphisms (SNPs) in promoter of IL-18 gene at the position-137G/C (RS187238) in patients with coronary artery disease, using the ligase detection reaction-polymerase chain reaction (LDR-PCR) method. Subjects studied in this research are Chinese Han population in Xiangfan region.Methods1 Patient:Cases of coronary artery disease and Control Cases were identified by coronary angiography. Assessment of coronary stenosis requires in at least two orthogonal projections. Lesion severity was then assessed in the projection revealing the greatest extent of narrowing. The diameter of the narrowed segment and reference vessel may be measured by digital and stenosis severity calculated as: %stenosis= (lumen diameter at stenosis site/reference diameter)×100. Diameter narrowing of more than 50% was considered to be Patients Group. The degree of severity is commonly classified into one of the following categories:(1), Normal coronary arteries; (2), Irregularities; (3),< 50% stenosis; (4),50%-69% stenosis; (5),70%-89% stenosis; (6),> or= 90% stenosis; (7), Occlusion. Categories (1), (2), and (3) were belonged to the Control Cases; Category 4) defined as Patients Group 1 (50%～69%stenosis); Category (5) defined as Patients Group 2 (70%～89% stenosis); Categories (6) and (7) were defined as Patients Group 3 (> or= 90% stenosis and Occlusion). Blood was collected from the arteriae femoralis for detection of the serum levels of IL-18 before selective coronary angiography. The concentration of serum IL-18, TNFaand IL-10 was measured with ELISA in patients with acute coronary syndrome (including unstable angina pectoris and acute myocardial infarction), patients with stable angina pectoris and control cases. The relations of the levels of serum interleukin-18, tumor necrosis factor-a and interleukin 10 between groups were analyzed.2 Patients with coronary heart disease were divided in two groups based on the presence or absence of type 2 diabetes mellitus. Diabetes mellitus was considered present if the fasting glucose level was at least 7 mmol/L, or if the patient had been informed of the diagnosis and treatment had been prescribed with a special diet, oral antidiabetic drugs or insulin. The concentration of serum IL-18 was measured with ELISA. The relations of the levels of serum interleukin-18 between groups were analyzed.3 Ultrasonography was performed to measure the carotid artery intima-media thickness and to observe the incidence rate of carotid atheromatous plaque in patients who had been examined by selective coronary angiography. The concentration of serum IL-18 was measured.4 The levels of serum IL-18 in patients with coronary artery disease before percutaneous coronary intervention (PCI) and 12 hours and 2 weeks after percutaneous coronary intervention therapy were measured, changes of IL-18 were analyzed. The cardiovascular events were followed up 12 months after therapy.5 The functional-137 G/C polymorphism (rs187238) in the promoter region of IL-18 gene was genotyped using the ligase detection reaction-polymerase chain reaction. Case patients of coronary artery disease and control patients were identified by coronary angiography and the plasma IL-18 concentrations were measured. Correlation between plasma IL-18 protein levels and genotypes of IL-18 polymorphism in studied population were analyzed.Results1 The serum levels of IL-18 and TNF-awere higher, and level of IL-10 was lower in than patients with coronary heart disease than those in control cases (P< 0.01). The levels of IL-18 and TNF-awere higher, and level of IL-10 was lower in acute coronary syndrome (including unstable angina pectoris and acute myocardial infarction) than patients with stable angina pectoris (P< 0.01). The level of IL-18 in Patients Group 2 (70%～90%stenosis) and Patients Group 3 (> 90% stenosis and Occlusion) was higher than Patients Group 1 (50%～70% stenosis) respectively. There were no significant differences in the levels of TNF-αand IL-10 between these groups.2 The coronary heart disease patients with type 2 diabetes mellitus were similar to the non-diabetic coronary heart disease patients with respect to age, BMI and lipid (P> 0.05). The serum level of IL-18 in patients with coronary heart disease was higher than that with control patients (P< 0.01). Concentration of IL-18 was higher in patients with diabetes mellitus than patients with non-diabetes mellitus. 3 Carotid artery intima-media thickness in coronary disease group was significantly greater than control group (0.93mm±0.26mm vs 0.65mm±0.13mm, P< 0.01). The incidence rate of an carotid atherosclerotic plaques in coronary disease group was significantly higher than control group (97.65% vs 6.9%, P< 0.01). The serum level of IL-18 was in coronary disease group was higher than control group (316.5 pg/ml±94.2 pg/ml vs 36.38 pg/ml±18.99 pg/ml, P< 0.01).4 The concentration of IL-18 12 hours after percutaneous coronary intervention therapy in patients with coronary artery disease was significantly increased than that before therapy (423.5 pg/ml±298.7 pg/ml vs.316.5 pg/ml±274.2 pg/ml, P<0.01), but 2 weeks later it was decreased significantly than that before therapy (286.7 pg/ml±256.9 pg/ml vs.316.5 pg/ml±274.2 pg/ml, P<0.01).5 A significant increase of G allele or GG-genotype was observed in 241 case patients compared to 145 control individuals (frequency of G allele= 0.90 vs 0.83, P=0.004; frequency of GG-genotype= 0.81 vs 0.68, P= 0.005). In case patients, G allele carriers in multi-vessel disease patients had a higher occurrence rate when compared to single-vessel disease patients, but no significant difference was detected (frequency of G allele= 0.92 vs 0.88, P=0.107; frequency of GG-genotype = 0.84 vs 0.75, P= 0.089). IL-18 protein concentration of the-137GG genotype was much higher than concentration of CG and CC genotype (case patients:229.1±131.5 vs 122.7±73.6 pg/ml, P< 0.001; control patients:65.9±31.6 vs 42.4±19.5 pg/ml, P< 0.001).ConclusionsIL-18 and TNF-a are suggested to play a role in atherogenesis and atheromatous plaque rupture leading to the coronary artery disease. Conversely, the anti-inflammatory cytokine IL-10 seems to have an atheroprotective role. Patients of type 2 diabetes mellitus predispose to a higher serum level of IL-18, which may explain their vulnerability to fatal cardiovascular events. Carotid artery intima-media thickness, the incidence rate of carotid atheromatous plaque and the serum levels of IL-18 can be used as a satisfactory index to quantitatively detect carotid artery atherosclerosis, and further to judge the risk factors of coronary heart disease. There is inflammation after percutaneous coronary intervention therapy. IL-18 are sensitive to reflect early inflammation and the changing degree of IL-18 may be one of predictors for cardiovascular events after coronary intervention therapy. IL-18 promoter-137G/C polymorphism influences IL-18 levels and the occurrence of coronary artery disease, suggesting that IL-18 is causally involved in the development of atherosclerosis. In summary, IL-18 and Coronary Artery Disease are closely related.更多还原