【作者】 李杰；

【导师】 袁肇凯；

【作者基本信息】 湖南中医药大学 ， 中医诊断学， 2006， 博士

【摘要】 第一部分冠心病血瘀证家系与ACE、FⅧ、ApoE、PAI-1基因多态性的连锁研究背景:CHD是一种多因素疾病,受环境和遗传等多因素的影响,为有明显遗传倾向的多基因病。CHD的发生与发展与多种易患基因多态性有关,尤其与血管紧张素转换酶插入/缺失(ACE I/D)基因多态性、载脂蛋白E(ApoE)基因多态性、凝血因子Ⅶ(FⅦ)基因多态性、纤溶酶原激活物抑制物-1(PAI-1)基因多态性等密切相关,它们通过调控脂质代谢、凝血功能和血管内皮功能而影响CHD临床表型。中医理论认为冠心病基本病机为心脉瘀阻,血瘀证是CHD最常见的证型,而ApoE、FⅦ、ACE、PAI-1基因多态性是否为CHD血瘀证家系的遗传分子生物学标志之一,值得深入研究。目的:了解湖南汉族家系人群ACE、FⅦ、Apoe、PAI-1基因各基因型分布频率,观察ACE基因(I/D)多态性、FⅦ基因(M1/M2)多态性、ApoE基因(ε2、ε3、ε4)多态性、PAI-1基因多态性与CHD血瘀证家系的关系及对CHD血瘀证易患影响。方法:本研究采用病例对照设计,筛选25个CHD血瘀证家系、15个CHD非血瘀证家系及10个健康人家系为研究对象。采用直接PCR和PCR-RFLP技术检测ACE基因I/D、ApoE基因(ε2、ε3、ε4)、FⅦ(M1/M2)基因多态性,用PCR-VNTR技术检测PAI-1基因多态性。用条件Logistic回归进行受累亲属对分析(APM)分析,将家系组成的APM对用单变量的条件Logistic回归以及调整传统危险因素后的多变量条件Logistic回归分析;在对FⅦ、ACE、ApoE、PAI-1基因多态与冠心病基因存在关联的基础上;利用核心家系资料进行单体型相对风险分析(HHRR),连锁不平衡检验(TDT)分析,检验该基因座是否与中国湖南汉族人群冠心病血瘀证基因存在连锁。结果:1.湖南汉族家系人群中:①ACE基因型分布在双亲中,DD 12.5 0%,ID 48.21%,Ⅱ39.29%;Ⅰ等位基因频率63.39%,D等位基因频率36.61%。在患者中,DD 14.29%,ID 53.57%,Ⅱ32.14%;Ⅰ等位基因频率58.93%,D等位基因频率41.07%。②FⅦ基因型分布在双亲中M1等位基因频率78.57%,患者的等位基因M1频率85.71%。③ApoE基因型分布在双亲中,E2/3 8.93%,E2/4 10.71%,E3/3 53.57%,E3/4 23.21%,E4/4 3.58%;等位基因ε2、ε3、ε4频率分别为9.82%、69.64%、20.54%;患者中,E2/314.29%,E2/4 7.14%,E3/3 67.86%,E 3/4 10.71%,E4/4 0%:ε2、ε3、ε4等位基因频率分别为10.71%、80.36%、8.93%。2.在单体型相对风险分析(HHRR)中,ACE的D基因与冠心病血瘀证不关联,FⅦ的M1基因和ApoE的ε4基因及PAI-1CA18基因多态性与冠心病血瘀证相关联。3.在连锁不平衡分析中,ACE的D等位基因和FⅥ的M1等位基因与冠心病血瘀证易患位点不连锁;ApoE的ε4等位基因和PAI-1的CA18等位基因与冠心病血瘀证易患位点连锁,可能为中国湖南汉族人群冠心病血瘀证的易患基因。4.在受累亲属对分析中,Ⅱ、ID、DD种基因型和M1M1、M1M2、M2M2种基因型在受累亲属对分析中分布无显著差别,PAI-1基因型CA18和ApoEε4基因型在血瘀证受累亲属对分析中分布有显著差别。结论:1.本项研究CHD家系ACE、FⅦ、ApoE、PAI-1基因的基因型、等位基因都没有偏离Hardy-weinberg平衡。2.ACE的D等位基因可能与冠心病血瘀证不关联,FⅦ的M1等位基因和ApoE的ε4等位基因及PAI-1 CA18等位基因可能与冠心病血瘀证相关联。3.ACE的D等位基因和FⅦ的M1等位基因可能不是中国湖南汉族人群冠心病血瘀证的易患基因;而ApoE等位基因ε4和PAI-1等位基因CA18与冠心病血瘀证易患位点连锁,可能为中国湖南汉族人群冠心病血瘀证的易患基因,且是CHD家系血瘀证独立的危险因素。4.冠心病血瘀证可能是一种具有遗传倾向的“多基因证”。第二部分冠心病血瘀证家系与脂质代谢、凝血功能及血管内皮功能相关性研究背景:已有的研究证明,CHD血瘀证的形成与体内脂质代谢紊乱、血管内皮损伤及凝血机制失常,血液处于血栓前高凝状态的病理生理过程相似,而且CHD的发病有家族聚集倾向,其发病率高于群体发病率。血脂紊乱是CHD发病与进展之重要危险因素之一,可作为中医辨证分型的一个客观指标;血瘀证患者存在着血栓前高凝和纤溶功能低下状态,其血栓形成是气滞血瘀和气虚血瘀的病理基础;血管内皮细胞内分泌功能失调可能是构成冠心病血瘀证的病理基础之一。中医认为冠心病基本病机为心脉瘀阻,临床和实验研究表明血瘀证是CHD最常见的证型之一。因此,脂质代谢紊乱、血管内皮损伤及凝血机制失常可能是CHD血瘀证家系的三个遗传易患特征。目的:1.分析CHD血瘀证家系患者脂质代谢、血管内皮功能及凝血机制相关指标,探讨这些指标与CHD血瘀证家系的遗传易患关系。2.观察家系中ACE基因I/D多态性对ET/NO的影响、FⅦ基因M1/M2多态性与血浆FⅦ活性(FⅦc)的关系,及PAI-1基因多态性对PAI-1活性的影响。方法:本研究采用病例对照设计,筛选25个CHD血瘀证家系、15个CHD非血瘀证家系患者及10个健康人家系为研究对象。采用硝酸还原酶法测定一氧化氮(NO),放免法测定内皮素(ET),血管紧张素Ⅱ(AgⅡ),发色底物法检测纤溶酶原激活剂抑制物活性(PAI)、抗凝血酶Ⅲ(ATⅢ)、组织型纤溶酶原激活剂(tPA),ELISA法测定血小板α颗粒膜蛋白(GMP-140),凝血因子Ⅶ活性(FⅦc)检测采用一期法进行检测。结果:1.CHD血瘀证家系组AgⅡ含量高于CHD非血瘀证家系组、健康对照组(p＜0.01或p＜0.05)。CHD血瘀证家系组ET含量、NO含量、NO/ET与健康对照组比较有显著性差异(p＜0.01),与CHD非血瘀证家系组相比差异无显著性意义(p＞0.05)。2.CHD血瘀证家系患者血浆ATⅢ浓度明显低于CHD非血瘀证家系组和健康对照组,GMP-140含量、PAI活性明显高于CHD非血瘀证家系组和健康对照组(p＜0.05～0.01),tPA的活性明显低于健康对照组(P＜0.01),与CHD非血瘀证家系组无差异。3.ACE基因I/D多态性对ET/NO的影响:CHD血瘀证家系组、CHD非血瘀家系组、健康对照组ACE基因3个基因型ET/NO值呈现Ⅱ→ID→DD的递增趋势。DD型比Ⅱ型ET/NO值显著增高(p＜0.0 5或＜0.01)。4.FⅦ基因多态性与血浆FⅦ活性(FⅦc)的关系:其中M1M1基因型的FⅦc含量呈CHD血瘀证家系组＞CHD非血瘀证家系组＞健康对照组呈递减趋势;而M1M2基因型相反。5.PAI-1基因多态性与PAI-1的活性关系:PAI-1的活性与CA拷贝数呈高度负相关,拷贝数越少,PAI-1活性越高;其中以血瘀证PAI-1活性更高,冠心病血瘀证家系组CA重复拷贝数N＜20的例数明显高于非血瘀证组。结论:1.冠心病血瘀证家系存在血管舒缩功能紊乱,表现为血管持续痉挛状态。2.冠心病血瘀证家系存在凝血、抗凝血及纤溶系统等功能紊乱,表现为血栓前高凝状态。3.血瘀证家系组ACE基因对ET/NO值表明DD型比Ⅱ型明显升高,可能是ACE引起冠状动脉痉挛的又一重要作用机制。4.血瘀证家系组FⅦ基因M1M1基因型与血浆FⅦ活性(FⅦc)密切相关。5.PAI-1基因的CA重复拷贝数在血瘀证家系中与血浆PAI-1活性呈负相关。6.冠心病家系血瘀证的形成与脂质代谢紊乱、凝血机制的异常、血管内皮细胞的损伤密切相关。更多还原

【Abstract】 Chapter 1 Association between Angiotensin-Converting Enzyme Gene、Coagulation FactorsⅦGene、apolipoprotein E(ApoE) gene and Plasminogen activator inhibitor-1 gene Polymorphisms with Blood Stasis Syndrome in Coronary Heart Disease PedigreesBackground:CHD was a multifactorial and multigenic disease and influenced by both environmental and genetic factors.During the produce and development of CHD,the insertion/deletion polymorphism of the Angiotensin converting enzyme(ACE)gene being proved to be related to the vessel endothelium functions,the apolipoprotein E(ApoE) gene being proved to be related to the mechanisms of serum lipids,coagulation factorsⅦ(FⅦ) gene being proved to be related to the function disorders of coagulation and Plasminogen activator inhibitor-1 gene Polymorphisms.they play important role.The heart blood stasis syndrome is one of the commonest syndrome,ApoE、FⅦ、ACE and PAI-1 gene polymorphism were probably the genetic marker of HBSS in CHDP.Objective:To learn the distributions of ACE、FⅦ、ApoE、PAI-1 in Hunan Province of China and to find the relationships between polymorphisms of ACE、FⅦApoE、PAI-1 gene with HBSS in CHDP and to study the effect of susceptibility with HBSS in CHDP.Methods:This was a case-control study,which enrolled 25 CHDP with HBSS,15 CHDP with non- HBSS and 10 pedigrees without CHD.PCR and PCR-RFLP techniques were performed to determine the genotypes of ACE、FⅦ、ApoE gene.PCR-VNTR techniques were performed to determine the genotypes of PAI-1 gene. Conditional Logistic regression was used to analyze the affected pedigree-member method(APM),which is analyzed by unvariable conditional Logistic regression and multiple Conditional Logistic regression after adjusting effects of traditional risk factors of CHD.After showing that the polymorphism of ACE、FⅦ、ApoE、PAI-1 gene are associated with CHD,the haplotype-based haploytpe relative risk(HHRR) and the transmission/disequilibrium test(TDT) are used to prove whether the gene is associated or linked with HBSS in CHD in Chinese Hunan han nation population.Results:1.In Hunan Province of China,the ACE I/D genotyped distribution was DD 12.50%、ID 12.50%andⅡ39.29%,respectively in parents and was DD 14.29%,ID 53.57%,Ⅱ32.14%,respectively in parents;Allele frequencies for D and I were 36.61%,63.39%,respectively in patients and D and I were 41.07%,58.93%.Allele frequencies of FⅦM1 gene were 78.57%in parents and were 85.71%in patients;the ApoE genotype distribution was E2/3 8.93%,E2/4 10.71%,E3/353.57%,E3/4 23.21%,E4/4 3.58%,respectively in parents and E2/3 14.29%,E2/4 7.14%,E3/3 67.86%,E3/4 10.71%,E4/4 0%, respectively in patients;Allele frequencies forε2、ε3、ε4 were 9.82%、69.64%、20.54%,respectively in parents and were 10.71%、80.36%、8.93%in patients.。2.In the haplotype-based haploytpe relative risk(HHRR),ACE gene D allele is not associated with HBSS in CHD;FⅦgene M1 allele、ApoE geneε4 allele and PAI-1 gene CA18 allele are associated with HBSS in CHD.3.In the transmission/disequilibrium test(TDT),ACE gene D allele and FⅦgene M1 allele are not linked with susceptibility location of HBSS in CHD.ApoE geneε4 allele and PAI-1 gene CA18 allele are linked with susceptibility location of HBSS in CHD,which might be associated or linked with HBSS in CHD in Chinese Hunan han nation population.4.In the affected pedigree-member method(APM),There were no difference in any genotype and allele frequencies of both ACE and FⅦgenes. There were significant difference in ApoE geneε4 allele and PAI-1 gene CA18 allele.Conclusions:1.There were no significant difference in genotype distribution and allele frequencies of ACE、FⅦ、ApoE、PAI-1 gene in Hunan people compared with Chinese people.2.ACE gene D allele is not associated with HBSS in CHD;FⅦgene M1 allele、ApoE geneε4 allele and PAI-1 gene CA18 allele are associated with HBSS in CHD.ApoE-ε4 allele was independent risk factor for the HBSS in CHD.3.ACE gene D allele and FⅦgene M1 allele are not linked with susceptibility location of HBSS in CHD.ApoE geneε4 allele and PAI-1 gene CA18 allele might be associated or linked with CHD with HBSS in Chinese population.ApoE-ε4 allele and PAI-1 gene CA18 were independent risk factor for the HBSS in CHD.4.CHD with HBSS was probably a multigenic syndrome with genetic factors.Chapter 2:The study of Association between the metaboly of serum lipid、the mechanisms of coagulation、vessel cell endothelial function and Blood Stasis Syndrome in Coronary Heart Disease PedigreesBackground:Research have proved the formation of HBSS in CHD is relate with the turbulence metaboly of serum lipid,the vessel endothelium damnification, the turbulence mechanisms of coagulation and is similar to the course of pathologic physiology that the blood is being the high coagulation before thrombus,the formation of HBSS in CHD have the family assembly tendency,in which incidence of a disease is higher than the colony incidence of a disease.turbulence metaboly of serum lipid is one of the important dangerous factors in CHD,which is used as a impersonality index in distinguishing syndrome and discriminate model.The high coagulation before thrombus and the low function of plasmin are found in patients of HBSS.The formation of thrombus is the pathologic base in sluggish gas to blood stasis syndrome and weak gas to blood stasis syndrome.The vessel endothelial dysfunction may be important mechanisms in the manifestation of CHD.The heart blood stasis syndrome is one of the commonest syndrome,the turbulence metaboly of serum lipid,the vessel endothelium damnification,the turbulence mechanisms of coagulation is probably three inheritance susceptibility characteristics of HBSS in CHD. Objective:1.To iunvestigate the index on the metaboly of serum lipid、the mechanisms of coagulation and the mechanism of endothelium dysfunction,and the inheritance susceptibility relations between this index and the heart blood stasis syndrome in coronary heart disease.2.To investigate the effect on the insertion/deletion polymorphism of ACE gene on ET/NO an,the polymorphism of FⅦon FⅦc activity level and the polymorphism of PAI-1 on PAI-1 activity level.Methods:This was a case-control study,which enrolled 25 CHDP with HBSS,15 CHDP with non- HBSS and 10 pedigrees without CHD.The levels of antithrombinⅢ(ATⅢ),GMP-140,tissue-type plasminogen activator(tPA), plasminogen activator inhibitor(PAl),plasma factorⅦcoagulant activity(FⅦc),angiotensinⅡ(AgⅡ),nitrogen monoxide(NO),endothelins(ET) were tested with radioimmunoassy and chemistry method.Results:1.The levels of AgⅡof HBSS group in CHDP were significantly higher than those of none-HBSS group in CHDP and healthy group(p＜0.05 or 0.01). The levels of ET,NO,NO/ET of HBSS group were significantly lowerer than those of healthy group(p＜0.05),but no significant difference compared with none-HBSS group in CHDP.2.The level of ATⅢof HBSS group in CHDP was significantly lowerer than that of healthy group(p＜0.05 or 0.01).The levels of GMP-140、PAI of HBSS group in CHDP were significantly higher than those of none-HBSS group in CHDP and healthy group(p＜0.05 or 0.01).There was significant difference between HBSS group in CHDP and healthy control group in activator of plasminogen(tPA),but no significant difference compared with none-HBSS group in CHDP.3.The effect on the insertion/deletion polymorphism of ACE gene on ET/NO:In every group,the ET/NO levels were associated with ACE I/D polymorphism:DD＞ID＞Ⅱ.There was significant difference in people carrying genotype DD compared with carrying genotypeⅡ(p＜0.05=.4.The effect on FⅦpolymorphism on levels of FⅦc activity.The levels of FⅦc activity in people carrying genotype M1M1:HBSS group in CHDP＞none-HBSS group in CHDP＞healthy control group;But contrary to genotype M1M2.5.The effect on PAI-1 polymorphism on levels of PAI-1 activity.There was correlation between the genotypes and the plasma PAI-1 activity.The shorter the dinucleotide repeats,the higher the level of PAI-1 activity.Expecially,the higher in HBSS in CHDP,the dinucleotide repeats under 20 of CA in HBSS in CHDP was higher than non-HBSS in CHDP. Conclusions:1.There was impaired endothelium dependent vascular diastolic-systolic function in HBSS of CHDP,being as impaired endothelium dependant vascular vasorelaxation function.2.There were the function disorders of coagulation,anti-coagulation and fibrinolysis in HBSS of CHDp,the state of excessive coagulation,and deficiency fibrinolysis before thrombosis.3.There was significant difference that carrying genotype DD compared with carrying genotypeⅡin ACE gene of HBSS of CHDP,which may be important action of coronary artery spasm.4.There was high correlation between the genotypes M1M1 in FⅦgene of HBSS of CHDP and levels of FⅦc activity.5.There was high negenative correlation between the genotypes and the level of plasma PAI-1 activity.6.HBSS in CHDP is relate with the turbulence metaboly of serum lipid, the vessel endothelium damnification,the turbulence mechanisms of coagulation.更多还原