【作者】 王丽萍；

【导师】 袁肇凯；

【作者基本信息】 湖南中医药大学 ， 中医诊断学， 2011， 博士

【摘要】 目的：探讨冠心病血瘀证遗传特征及分析体质对冠心病血瘀证的影响。方法：1.文献研究：本文通过在维普、中国知网、万方数据库、PubMed.Circulation、Refdoc.fr等资源网中检索"冠心病.血瘀证.遗传流行病学、体质、CHD.Gene.Gene chip"等词查找关于冠心病、冠心病血瘀证遗传特征的文献,对文献逐一查找,归纳总结,同时在了解冠心病血瘀证的遗传特征研究现状,并结合参研古今体质文献,了解体质的研究进展及体质对冠心病血瘀证的影响研究。2.遗传流行病学研究：以遗传流行病学方法,采用病例对照研究。通过家族聚集性分析、分离比、遗传度并对多基因遗传方式确认及相关性分析,对冠心病血瘀证的遗传特征及体质进行研究。3.基因组学研究：采用Affymetrix HumanGenome U133 Plus 2.0基因芯片对家系冠心病血瘀证、家系冠心病非血瘀证、家系非冠心病血瘀证、家系健康人、非家系冠心病血瘀证、非家系健康人进行研究,筛选观察冠心病血瘀证差异基因表达谱。通过基因本体论(Gene Ontology, GO)分析,核准每个差异基因的分子功能、通过通过http://www.biocarta.com/及http://www.genome.jp/kegg/网站找到每一个差异基因所在通路,并利用超几何分布统计学方法分析通路结果,通过P值(P<0.05)判断pathway显著性,筛选有意义的目标通路。,结果:1.文献研究：现阶段的冠心病血瘀证遗传特征的研究主要以研究基因多肽性为主,并总结归纳出了冠心病的可能候选基因：(1)脂质代谢：载脂蛋白家族、ABCA家族、固醇调节元件结合蛋白家族、LDL及氧化型、LDL受体、HDL受体、C5L2、脂蛋白、糜酶、清道夫受体、CD36受体基因多态性；(2)炎症：C反应蛋白、肿瘤坏死因子、白细胞介素、补体家族、髓过氧化物酶、P物质、血浆淀粉样蛋白A、Toll样受体、CD40及其受体、选择素、整合素、白三烯、热休克蛋白、血小板活化因子、基质金属蛋白酶、转化生长因子、Fas及其配体；(3)凝血及纤溶系统：凝血因子、血小板受体、纤维蛋白原、血管性假血友病纤溶酶原、ADP受体、胶原受体、血栓素合酶、环氧化酶、前列环素合酶、血栓受体、前列环素受体(4)内皮系统：内皮素及其受体、一氧化氮合酶及其受体、V因子、血栓调节蛋白、内皮衍化生长因子、G蛋白、EDFIF、肝细胞生长因子、胎盘生长因子、妊娠相关蛋白A;(5)肾素-血管紧张素-醛固酮系统：肾素受体、血管紧张素转换酶、醛固酮受体、血管紧张素受体(6)其他：血红素甲酶、Kalirin VAMP, CYP家族、肌细胞特异性增强因子2。同时,对体质学说的概念、体质与证候的关系、体质与遗传的关心发现在冠心病血瘀证的研究中引入体质学说有利于以指导冠心病血瘀证的预防、诊断和治疗,以有利于阐释血瘀证的遗传特征。2.遗传流行病学研究：①通过对观察组与对照组家系一、二级亲属冠心病血瘀证患病率的研究,结果显示存在显著性差异(p<0.05),同时在冠心病血瘀证患者的家族分析中,证实冠心病血瘀证确实表现出家族聚集性的特点。这些研究结论都提示：冠心病血瘀证发病具有遗传倾向,即遗传因素在冠心病血瘀证的发生过程中起着一定的作用；通过估算冠心病血瘀证分离比及遗传度得出：冠心病血瘀证的分离比为0.1296(0.1020-0.1572),表明冠心病血瘀证属多基因遗传病,经Falconer法估算一、二级亲属遗传度分别为58.04%、54.80%,属于中度遗传；采用Penrose’s法估计遗传模式,冠心病血瘀证组s/q=3.47,较接近1/(?)=8.80;通过对冠心病血瘀证多基因遗传方式确认,结果显示：当一级亲属遗传度为0.5804时,采用江三多阈值模型理论推算预期值,其公式为Xr=Xg-rh2ag,结果冠心病血瘀证病一级亲属的理论发病率为8.00%,与实际发病率5.89%呈接近趋势,说明冠心病血瘀证在一级亲属中的分布符合多基因遗传的特征。同时冠心病血瘀证病二级亲属的遗传度为0.5480时,其理论发病率为6.20%,与实际发病率4.13%呈接近趋势,说明冠心病血瘀证在二级亲属中的分布符合多基因遗传的特征。其次,冠心病血瘀证于两组未见男女性别差异。②冠心病血瘀证的中医体质特征：冠心病血瘀证患者的主体质以阳虚质、痰湿质、阴虚质为主；兼体质是以痰湿质、阳虚质、阴虚质为主。九种体质的遗传度均低于50%,因此在冠心病血瘀证家系的体质中环境的影响大于遗传的影响。也就是说,体质可以作为影响冠心病血瘀证的一个相关因子,但对于冠心病血瘀证的遗传并不起决定作用。这与临床是相符的,在临床中冠心病血瘀证的发展与不良的生活习惯及饮食等有密切关系。③在对观察组进行体质的相关性分析发现冠心病血瘀证的发病与主体质存在着相关性(p<0.05)。对冠心病血瘀证遗传度研究,结果提示：九种体质的遗传度均低于50%,因此在冠心病血瘀证家系的体质中环境的影响大于遗传的影响。体质可以作为影响冠心病血瘀证的一个相关因子,但对于冠心病血瘀证的遗传并不起决定作用。这与临床是相符的,在临床中冠心病血瘀证的发展与不良的生活习惯及饮食等有密切关系。3.基因组学研究：结果显示冠心病血瘀证差异基因主要以炎症因子为主。当p<0.05时研究发现,冠心病血瘀证的差异基因表达主要涉及到如下几类基因系的表达改变：①趋化因子②白介素细胞因子③基质金属蛋白酶MMPs系；④成纤维细胞生长因子⑤细胞色素氧化酶P450(CYP)系;⑥与缺氧相关的基因。经BioCarta及KEGG通路分析冠心病血瘀证的遗传相关差异基因,进一步验证冠心病血瘀证主要涉及到炎症、斑块形成、内皮损伤方面。在冠心病血瘀证痰湿质患者的体内发现与水通调有关的AQP4基因表达发生上调。结论：1.强调“辨体质”、“辨证”等多种诊断分析方法综合运用,有利于对冠心病血瘀证本质的全面认识。2.冠心病血瘀证发病具有遗传倾向,即遗传因素在冠心病血瘀证的发生过程中起着一定的作用。冠心病血瘀证属多基因遗传病,亲属遗传度属于中度遗传。3.冠心病血瘀证主体质主要是以阳虚质、痰湿质、阴虚质为主兼体质是以痰湿质、阳虚质、阴虚质为主。体质与冠心病血瘀证存在着相关性。4.冠心病血瘀证的差异基因表达主要涉及①趋化因子②白介素细胞因子③基质金属蛋白酶MMPs系;④成纤维细胞生长因子⑤细胞色素氧化酶P450(CYP)系；⑥与缺氧相关的基因。5.经BioCarta及KEGG通路分析冠心病血瘀证的遗传相关差异基因,进一步验证冠心病血瘀证主要涉及到炎症、斑块形成、内皮损伤方面。更多还原

【Abstract】 Objective:Discussed Genetic Characteristics of blood stasis of CHD and analyzed physical effects of blood stasis of CHDMethod:1.Document Research:This article Searched the words"coronary heart disease, blood stasis, genetic epidemiology, physical, CHD, Gene, Gene chip" to finding Document of blood stasis of CHD and summarized this Document to understand the Research that genetic characteristics of CHD and affect physical effects of blood stasis of CHD in the VIP, CNKI, Wanfang database, PubMed, Circulation, Refdoc.fr network resources2.Physical and genetic epidemiological studies:Research Methods that blood stasis of CHD is family aggregation analysis, segregation ratio, heritability, Genetic approach by Genetic epidemiological methods, case-control study3.Gene chip technology:Using Affymetrix HumanGenome U133 Plus 2.0 gene chip CHD on the family, family, non-blood stasis syndrome with coronary heart disease, family, non-blood stasis syndrome with CHD, family health, non-family CHD, non-family study healthy people, CHD Screening gene expression profiles observed. By Gene Ontology (Gene Ontology, GO) analysis of differentially expressed genes for each approved the molecular function, through the website by http://www.biocarta.com/and http://www.genome.jp/kegg/find each Where the pathway between genes and used the hypergeometric distribution of the results of statistical analysis channel, through the P value (P<0.05) determine the pathway was significant, meaningful goals screening pathway. Results:1.Document Research:CHD at this stage of the study the genetic characteristics of the study of gene peptide-based, and summarizes the potential candidate gene for CHD. Possible candidate genes with CHD (1) lipid metabolism:apolipoprotein family, ABCA family, sterol regulatory element binding protein family, LDL and oxidized, LDL receptor, HDL receptor, C5L2, lipoprotein, chymase, Scavenger receptor, CD36 receptor gene polymorphism; (2) inflammation:C reactive protein, tumor necrosis factor, interleukin, complement the family, myeloperoxidase, P substance, plasma amyloid A, Toll-like Receptor, CD40 and its receptor, selectin, integrin, leukotrienes, heat shock protein, platelet activating factor, matrix metalloproteinases, transforming growth factor, Fas ligand; (3) coagulation and fibrinolytic systems:Coagulation factors, platelet receptor, fibrinogen, plasminogen and von Willebrand, ADP receptor, collagen receptor, thromboxane synthase, cyclooxygenase, prostacyclin synthase, thromboxane receptors, prostaglandin PGI receptor (4) endothelial system:endothelin and its receptor, nitric oxide synthase and its receptor, V factor, thrombomodulin, endothelial derived growth factor, G protein, EDFIF, hepatocyte growth factor, placenta Growth factor, pregnancy associated plasma protein A; (5) renin-angiotensin aldosterone system:renin receptor, angiotensin converting enzyme, aldosterone receptor, angiotensin receptor (6) Other:A enzyme heme Kalirin VAMP, CYP family, the muscle cell-specific enhancer factor 2. The concept of physical theory, the relationship between physical fitness and syndromes, physical and genetic concerns found in the study of CHD in favor the introduction of physical theory to guide the CHD prevention, diagnosis and treatment in order to facilitate Interpretation of the genetic characteristics of blood stasis.2.Physical and genetic epidemiological studies:CHD①by case group and control group, one family, two relatives of the prevalence of CHD, the results showed significant differences (p<0.05), while patients in the family of CHD analysis confirmed that CHD does show the characteristics of familial aggregation. Conclusions of these studies suggest:CHD with a genetic tendency to blood stasis, that genetic factors in the occurrence of CHD play a role in the process; by estimating the segregation ratio and CHD Heritability obtained:Isolation of CHD ratio of 0.1296 (0.1020-0.1572), indicating that the Coronary Heart Disease is a multi-gene genetic diseases, by Falconer Estimating first and second degree relatives were 58.04% of genetic,54.80%, is moderate genetic; By Penrose’s method to estimate the genetic model, coronary heart disease and blood stasis syndrome group s/q=3.47, closer to 1/q=8.80; CHD by way of multiple genetic confirmation, the results show:when a relative degree of genetic 0.5804, the threshold model by Jiang Sanduo expected theoretical calculations, the formula for Xr=Xg-rh2ag, the results CHD incidence of disease theory of first-degree relatives was 8.00%, and the actual incidence rate of 5.89% was close to trend, indicating that relatives of CHD in the distribution in a more consistent genetic characteristics. Meanwhile relatives of CHD The heritability of disease secondary to 0.5480, its theoretical incidence rate of 6.20%, and the actual incidence rate of 4.13% was close to trend, indicating that relatives of CHD in the secondary distribution line with multi-genetic characteristics. Secondly, no-CHD gender differences in the two groups.①physical characteristics of Chinese medicine:Patients with CHD yang quality to the main body, phlegm, yin deficiency based; and physical fitness are phlegm, Yang and quality, yin deficiency based; Nine Constitutional heritability were less than 50%, the blood stasis of coronary heart disease in the family’s physical environment is greater than genetic influences. In other words, physical impact of Coronary Heart Disease as a relevant factor, but for Coronary Heart Disease does not play a decisive role of genetic. This is consistent with the clinical, Coronary Heart Disease in clinical development and bad habits and diet are closely related.③In the case of groups the correlation of physical stasis that the incidence of CHD with the main body there was a correlation (p<0.05). Heritability of CHD study, results suggest that:the genetic constitution of nine were less than 50% of the degree, so in Coronary Heart Disease in the family’s physical environment is greater than genetic influences. Physical impact of CHD as a relevant factor, but for CHD does not play a decisive role of genetic. This is consistent with the clinical, CHD in clinical development and bad habits and diet are closely related.3.Gene chip technology:The results showed that differences in gene CHD based mainly on inflammatory factors. When p<0.05 when the study found differences in CHD gene expression mainly related to the following types of gene expression changes in the Department:①Chemokine;②Cytokine interleukin;③Department of matrix metalloproteinases MMPs;④Fibroblast growth factor;⑤cytochrome oxidase P450 (CYP) system;⑥hypoxia-related Genes. The BioCarta and KEGG pathway analysis of CHD-related differences in the genetic gene, to further verify the Coronary Heart Disease mainly related to inflammation and plaque formation, endothelial damage aspects. Department of CHD at home, it found in Phlegm with water and adjusting the AQP4 regulation in gene expression.Conclusion:1. Which emphasis on "Distinguishing physical", "syndrome"and many other integrated use of diagnostic analysis is use for CHD comprehensive understanding of the nature2. CHD with a genetic tendency to blood stasis, that genetic factors in the occurrence of CHD play a role in the process. Coronary Heart Disease is a multi-gene genetic diseases, the relationship is moderate genetic heritability.3. CHD is the main body Main yang quality, phlegm, yin deficiency-based, and physical fitness is phlegm, Yang nature, yin deficiency based. CHD Constitution and there was a correlation.4. CHD mainly related to differences in gene expression①Chemokine;②Cytokine interleukin;③Department of matrix metalloproteinases MMPs;④Fibroblast growth factor;⑤cytochrome oxidase P450 (CYP) system;⑥hypoxia-related Genes.5. The BioCarta and KEGG pathway analysis of CHD-related differences in the genetic gene, to further verify the CHD mainly related to inflammation and plaque formation, endothelial damage aspects.更多还原