摘要
研究背景和目的:冠心病的发生是多种危险因素长期作用的结果所导致的。对冠心病的研究,是当前心血管领域里研究的焦点。血管内皮细胞功能失调几乎贯穿于各个冠心病发病学说之中,被广泛认为是冠心病发病进程中最重要的始动环节。人体内多种因素均不可避免地造成内皮细胞损伤。而人体内存在的内皮损伤后修复机制在防止各种危险因素累积以及各种病理效应叠加的过程中发挥着重要作用。血管内皮细胞生长因子及其受体系统在血管生成和内皮损伤修复过程中至关重要。VEGF与受体结合后,诱导血管内皮细胞的分裂增殖,从而发挥VEGF的生物学活性。KDR是2型VEGF受体,主要表达于内皮细胞,介导VEGF的生物学效应,在血管生成和修复中起了关键作用。VEGF及KDR在动脉粥样硬化斑块中高表达,并与斑块破裂有关,参与了冠状动脉粥样硬化过程。已有数据库显示KDR基因含有多个SNP位点,生物信息学提示某些SNP位点可能影响蛋白分子VEGFR2的表达情况及功能。本研究中,我们推测KDR基因启动子区天然存在的多态性位点SNP-604以及编码区多态性位点SNP1192和SNP1719可能与冠心病的个体遗传易感性有关。
研究方法:我们采用病例对照研究的方法分析了KDR基因-604SNP位点。其中包括两个独立的病例-对照人群:665例冠心病患者(191例急性冠脉综合征和474例稳定型冠心病)和1013例正常对照;369例冠心病患者和625例正常对照。基因分型采用PCR结合限制性内切酶消化的方法,并通过抽样测序验证。同时我们通过荧光素酶报告系统研究ELISA方法检测血清VEGFR水平验证了启动子区SNP-604对启动子活性的影响,利用凝胶阻滞电泳分析(EMSA)证明了启动子区SNP-604位点附近序列与核蛋白(推测含一种以上转录因子)结合,另外我们构建了编码区SNP位点突变型和野生型载体,通过配体-受体结合试验证明了编码区2个SNP位点影响VGEFR2与VEGF的结合效率。
研究结果:实验结果显示这3个SNP位点均与冠心病风险相关,在第一个病例-对照人群中SNP-604的OR=1.37(p=0.006),SNP1192的OR=1.41(p=0.011),SNP1719的OR=1.37(p=0.007),在第二个病例-对照人群中SNP-604的OR=1.40(p=0.015),SNP1192的OR=1.75(p=0.003),SNP1719的OR=1.50(p=0.010)。此外,功能学研究显示SNP-604降低了KDR基因启动子活性,其中等位基因为C基因型的KDR启动子活性比T基因型低68%,同时ELISA结果显示该SNP可以显著影响血清学KDR蛋白水平。凝胶迁移试验(EMSA)结果显示SNP-604位多态所在区域能与核蛋白结合,而SNP1192和SNP1719则可显著影响KDR蛋白与VEGF的结合效率。
结论:KDR基因位于启动子区的SNP-604和位于编码区的SNP1192、1719通过影响KDR的表达以及KDR与VEGF的结合效率,从而与冠心病遗传易感性相关。
Background and Objectives:Vascular endothelial growth factor(VEGF) and its receptor KDR(kinase insert domain-containing receptor/fetal liver kinase-1,also called VEGFR2) play critical roles in angiogenesis and vascular repair,which are involved in the progress of coronary heart disease. Our purpose was to determine whether the common polymorphisms (SNP-604,SNP1192,and SNP1719) in KDR are associated with risk of coronary heart disease.
Methods:The association of the 3 polymorphisms with risk of coronary heart disease was determined in 2 independent case-control studies:one comprised of 665 patients with coronary heart disease and 1,015 control subjects,and the other comprised of 369 patients and 625 control subjects.The SNP functions of KDR gene were studied by using luciferase reporter assays, determination of serum levels of KDR,and ligand-binding assays.
Results:The 2 independent population studies showed that the 3 polymorphisms were associated with risk of coronary heart disease with odds ratios of 1.37 for SNP-604(p=0.006),1.41 for SNP1192(p=0.011),and 1.37 for SNP1719(p=0.007) in the first population,and 1.40 for SNP-604 (p=0.015),1.75 for SNP1192(p=0.003),and 1.50 for SNP1719(p= 0.010) in the second population.The SNP-604C-bearing KDR promoter exhibited 68%of lower transcription activity than the SNP-604T-bearing promoter.The SNP 1192 and SNP1719 could obviously influence the efficiency of VEGF binding to KDR.
Conclusions:The KDR polymorphisms may serve as novel genetic markers for the risk of coronary heart disease.
引文
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[1]Keil U.Coronary artery disease:the role of lipids,hypertension and smoking.Basic R es Cardiol,2000,95(Suppl 11):152-158.
[2]韩淑芬.冠心病危险因素调查.中国慢性病预防与控制,2001,9(9):232.
[3]王抒,李健斋。冠心病危险因素在老年人中的分布和集聚[J].中华老年医学杂志,2004,23:157-159。
[4]刘平,等。1662名知识分子心血管病主要危险因素的现况研究[J].疾病控制杂志,2006,10:419-420。
[5]王薇,赵冬.我国心血管病及其主要危险因素的流行病学研究[J].首都医科大学学报,2005,26:143-1460.
[6]岳寒,顾东风,吴锡桂,等。首都钢铁公司5137名男工心肌梗死发病危险因素的研究.中华预防医学杂志,2004,38(1):43-46.
[7]Loomans CJ,De Koning EJ,Staal FJ,et al.Endothelial progenitor cell dysfunction in type 1 diabetes:another consequence of oxidative stress?[J].Antioxid Redox Signal,2005,7(11-12):1468
[8]Reinhart K,Bayer O,Brunkhorst F,et al.Markers of endothelial damage in organ dysfunction and sepsis[J].Crit Care Med,2002,30(5):302
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