【作者】 金国栋；

【导师】 徐耕； James Cameron；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 第一部分凝血因子ⅩⅢ-A Val34Leu基因多态性与冠心病、心肌梗死关系的研究研究背景目前凝血因子ⅩⅢ的Val34LCu基因多态性与冠心病、心肌梗死的关系研究报道仍存在许多争议,同时在亚洲人群中的研究较少。本文假设凝血因子ⅩⅢ-A亚单位Val34Leu基因多态性在中国汉族人群中存在,运用聚合酶链反应-单链核苷酸多态性(PCR-SSCP)的分子生物学方法,测定凝血因子ⅩⅢ的Val34Leu基因多态性,从而评价其在冠心病与心肌梗死中的作用。研究内容:本文调查了195例冠脉造影证实的冠心病患者与203例正常对照组的测定凝血因子ⅩⅢ的Val34Leu基因多态性。本研究中在所有冠心病患者与正常对照组人群中我们没有发现Leu/Leu基因型.Val34Leu基因多态性在冠心病与正常对照组的分布没有显著性差异(P=0.923)。亚组分析中,把冠心病组患者根据有无心肌梗死再分为2组,统计分析发现Val/Leu基因型与心肌梗死的降低有关(P=0.005;校正odds ratio=1.75;95%可信区间=1.28-2.25)。同时,研究发现中国汉族人群中Leu34等位基因低于西方白种人群。结论与创新:本研究初步研究证实了中国汉族人群中存在凝血因子ⅩⅢVal34Leu基因多态性,但其分布频率低于西方白种人群。研究结果也加强了凝血因子Leu34等位基因是心肌梗死保护因子的可能性。同时凝血因子ⅩⅢVal34Leu基因多态性也许对心肌梗死后心肌愈合产生影响,而且存在其它的凝血因子ⅩⅢ的基因多态性。进一步的大规模临床研究与多中心的队列前瞻性研究应更注重于心肌梗死后心肌愈合过程的病理生理机制。第二部分血小板ADP受体P2Y12 H1/H2基因多态性与冠心病的研究研究背景:血小板在动脉粥样硬化斑块破裂后的血栓形成中起着重要的关键作用。斑块破裂与急性冠脉综合征(acute coronary syndromes,ACS)或冠脉介入手术(percutaneous coronary interventions,PCI)治疗的患者有密切关系。在多种血小板激活的媒介物质中,二磷酸腺苷(adenosine diphosphate,ADP)尤为重要。二磷酸腺苷与血小板膜上相关受体结合,是凝血与血栓过程的重要中间物质。血小板激活后,二磷酸腺苷不仅从细胞类释放出来,而且可以激活血小板再活化,扩增整个血栓形成的过程。血小板膜上有2个主要受体:1:GTP(guanosine triphosphate)偶联蛋白受体,也就是G-蛋白结合位点;2:铁通道受体(ligand-gated ion channel)2 R.F.Storey,L.J.Newby and S.Heptinstall,Effects of P2Y(1)and P2Y(12)receptor antagonists onplatelet aggregation induced by different agonists in human whole blood,Platelets 12(2001),后者受体称为P2X1,而前者称为P2Y。这2个受体在血小板激活与聚集过程中都起着重要的作用。最近有研究报道在一组健康人群的实验中,血小板膜上P2Y12受体基因多态性被认为与血小板聚集率相关,而且与外周动脉疾病有关。本文测定了中国汉族人群中P2Y12受体的H1H2基因多态性的频率及与冠心病的关系。研究内容:观察冠状动脉造影患者血小板膜ADP受体P2Y12功能获得性基因多态性在分布及其与冠心病的关系,并探求该位点的突变与凝血酶的关系。应用聚合酶链反应和限制性内切酶片段长度多态性技术检测了654例进行选择性冠状动脉造影患者的血小板膜上ADP受体P2Y12 H1/H2基因型,其中302例CAD患者和352例对照组,结合造影结果探讨该基因多态性和冠心病的关系。ADP受体P2Y12 H1/H2基因多态性在本组人群中的分布频率为H1/H1 78.6%,H1/H2 19.7%,H2/H2 1.7%,等位基因为H1 88.5%,H2 11.5%.冠心病组为H1/H1223(73.8%),H1/H2 71(23.5%),H2/H2 8(2.7%),对照组为H1/H1 291(82.7%),H1/H2 58(16.5%)H2/H2 3(0.8%)基因型频率符合Hardy-Weinberg平衡定律,两者之间差异有显著性(χ2=8.808,P＜0.05)。等位基因为冠心病组H2 87(14.4%),H1517(85.6%),对照组H2 64(9.0%)H1640(90.1%),两者差异有显著性(χ2=8.987,P＜0.01).凝血酶中只有激活的部分凝血酶时间(APTT)与该位点的基因型有关。结论与创新:中国南方汉族人群中存在着ADP受体P2Y12 H1/H2的基因多态性,并且该多态性可能与冠心病的发生有关。更多还原

【Abstract】 PartⅠCoagulation FactorⅩⅢ-A Val34Leu Polymorphism and the Risk of Coronary Artery Disease and Myocardial Infarction in A Chinese Han PopulationBackgroundThere are controversial data regarding the impact of coagulation factorⅩⅢ-A subunit（FⅩⅢ-A） Val34Leu polymorphism in the pathogeneric of coronary artery disease（CAD） and myocardial infarction（MI）.Assuming this genetic factor is associated with the thrombotic process,we explored the role of FⅩⅢ-A Val34Leu in CAD and MI in a Chinese Han population.Methods and ResultsWe recruited 195 consecutive CAD patients confirmed by coronary angiography as well as a group of 203 controls.FⅩⅢ-A Val34Leu polymorphism was determined through polymerase chain reaction-single strand conformational polymorphism analysis.We did not find the Leu/Leu genotype in CAD patients or controls.No significant difference in Val34Leu gene polymorphism distribution was found between CAD patients and controls（P=0.923）.Subgroup analysis according to history of MI showed the heterozygote Val/Leu genotype was associated with a significantly decreased risk of MI（P=0.005;adjusted odds ratio=1.75;95%confidence interval =1.28-2.25）.Furthermore,our study displayed that the frequency of the Leu34 allele in a Chinese Han population was lower than that in Caucasian populations（2.5 vs. 20.4-28.3%）.ConclusionOur preliminary data indicates that the FⅩⅢ-A Leu34 allele may contribute a protective effect against the development of MI.There is a low prevalence of the Leu34 allele in Han Chinese compared to Caucasians. PARTⅡThe Association of ADP P2Y₁₂ H1/H2 Polymorphism and Coronary Artery DiseaseBackgroundPlatelets play a key role in the pathophysiology of thrombosis after plaque rupture. Plaque rupture occurs spontaneously in patients with acute coronary syndromes（ACS）, or may be induced in patients undergoing percutaneous coronary interventions（PCI）.Among the multiple mediators of platelet activation,adenosine diphosphate（ADP） plays a pivotal role.Adenosine diphosphate binds to several receptors on the platelet membrane.Adenosine diphosphate is one of the most important mediators of both physiological hemostasis and thrombosis.After platelet activation,ADP is not only released from its intracellular storage granules but also further activates platelets, amplifying this process.There are 2 main purinergic receptor types in the membrane:the guanosine triphosphate（GTP）-coupled protein receptors,known as G-protein binding sites,and the ligand-gated ion channel.The latter receptor is designated P2X₁ and the former is designated as P2Y,and each play a specific and complimentary role in platelet activation and aggregation.Recently,polymorphisms of the P2Y12 receptor have been associated with different degrees of platelet aggregation in healthy volunteers and also have been indicated to relate to artery disease.We investigate the P2Y12 receptor H1H2 gene polymorphism’s frequency distribution and the association with coronary artery disease. Methods and ResultsTo observe the frequencies of the polymorphism of Adenosine Diphosphate（ADP） Receptor P2Y12 H1/H2 Haplotype in Chinese and the relationship between the polymorphism and coronary artery disease（CAD）.ADP receptor P2Y12 H1/H2 Haplotype polymorphism was performed in 654 patients who were performed selective coronary angiography by using PCR-RFLP methods.The patients were classfied as 302 CAD patients group and 352 controls group.It was observed the relationship between the polymorphism of ADP receptor P2Y12 H1/H2 Haplotype polymorphism.The frequencies of genotype were H1/H1 78.6%,H1/H2 19.7%,H2/H2 1.7%.The frequencies of allele was H1 88.5%,H2 11.5%.The genotype distribution was in accordance with Hardy- Weinberg equilibrilum.A significant increase was found for the ADP receptor P2Y12 H1/H2 in CAD patients compared with controls（26.2%vs17.3%,χ2=8.808,P＜0.05）。A significant increase was found for ADP receptor P2Y12 H1/H2 Haplotype H2 allele in CAD patients compared with controls（14.4%vs9.0%,χ2=8.987,P＜0.01）.Only the relationship of the APTT and the genotype were observed in this study.ConclusionThe polymorphism of ADP receptor P2Y12 H1/H2 Haplotype is present in Chinese and the H2 allele is a major risk factor for CAD patients.更多还原