冠心病血瘀证的异质性研究
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摘要
[目的]
通过对冠心病血瘀证的表征(包括症状体征与血栓前状态分子)、基因表达谱和易感基因启动子甲基化状态的研究,探讨冠心病血瘀证的证本质是否存在着异质性特征。
[方法]
1.文献研究：通过检索中国知网(CNKI)、维普(CQVIP)、万方(WANFANG DATA)、中国生物医学文献数据库(CBM)及Pubmed等数据库,查询有关冠心病血瘀证的临床文献,主要集中在两个方面：①血府逐瘀汤治疗冠心病血瘀证的临床疗效；②冠心病血瘀证的客观化研究,包含采用高通量技术(基因组、蛋白质组、代谢组)及其他微观指标的文献。提取纳入文献的有关信息,采用meta分析等方法进行系统评价
2.临床研究：采用系统聚类分析等方法,以组间间距大、组内间距小的原则,对冠心病血瘀证患者的临床表征---症状体征与血栓前状态分子进行分析。
3.实验研究：应用基因表达谱芯片技术对研究各组(家系组：冠心病血瘀证、冠心病非血瘀证、非冠心病血瘀证、健康人；非家系组：冠心病血瘀证、健康人)的基因表达情况进行分析；采用实时荧光定量RT-PCR技术对挑选的若干差异表达基因进行验证；采用甲基化特异性PCR技术(MS-PCR)对若干基因启动子的甲基化状态进行分析。
[结果]
1.对纳入的近十年(2003-2012年)有关血府逐瘀汤治疗冠心病心绞痛血瘀证的18篇文献进行分析,结果表明血府逐瘀汤在改善中医证候疗效、心绞痛疗效和心电图疗效方面均有较好表现,但各独立研究内部在疗效程度上存在痊愈、有效、无效和加重的异质性。
2.各数据库获取的冠心病血瘀证高通量研究的文献共21篇(基因表达谱研究3篇,蛋白质谱研究11篇,代谢组学研究6篇),其他微观指标的研究35篇(外周血白细胞DNA单核苷酸多态性研究13篇,其他22篇)。其中对符合纳入标准的ACE、ApoE和FⅦ基因多态性研究、其他微观指标研究(一氧化氮、血管性血友病因子、内皮素、可溶性细胞间黏附分子、组织纤维蛋白溶解酶原激活物抑制剂-1、组织型纤溶酶原激活物)进行了meta分析,结果表明不管是高通量技术研究还是还原分析方法的研究,现有的各研究在结局指标间均存在不同程度的异质性。
3.对纳入的冠心病血瘀证患者66例(其中家系冠心病血瘀证患者39例,非家系冠心病血瘀证患者27例),以血瘀证的10类诊断要素胸闷、针刺痛、面紫、唇青、甲青、舌色、舌瘀斑、脉涩、脉结、脉代的评分结果进行症状体征的系统聚类分析(Ward法),结果表明,冠心病血瘀证的临床表征可以分为两个亚类,聚类1特征：以(胸闷+唇青+面紫)的症状组合为主要表现,其他症状体征少见；聚类2特征：以(胸闷+唇青+面紫)+(刺痛+舌色)的症状组合为常见症状,且舌瘀斑为极少见症状。通过统计学分析这2亚类分别与具有遗传背景的家系冠心病血瘀证和无遗传背景的非家系冠心病血瘀证有相关性,聚类1中家系冠心病血瘀证患者：非家系冠心病血瘀证患者为12/21(人),聚类2中非家系冠心病血瘀证患者：家系冠心病血瘀证患者为27/6(人),具有统计学意义。
以冠心病血瘀证患者的外周血血栓前状态分子(PTS)的8个标记物：血浆血栓调节蛋白(TM)、血小板表面P-选择素(P-Selectin)、凝血酶原片段1、2(F1+2)、可溶性纤维蛋白单体(SFMC)、组织型纤溶酶原激活物(tPA)、纤溶酶原激活物抑制物(PAI-1)、凝血酶-抗凝血酶Ⅲ复合物(TAT)和蛋白质C肽(PCP)为对象进行系统聚类和统计学分析。结果表明家系冠心病血瘀证与家系健康人比较在TN、P-Selectiin、F1+2、t-PA、TAT、PCP六个分子上有统计学差异,非家系冠心病血瘀证和非家系健康人间无统计学差异,而家系冠心病血瘀证与非家系冠心病血瘀证间在t-PA、PAI-1、TAT三个分子上有统计学差异。系统聚类分析(Ward法)表明PTS分子可以聚为两类,聚类1以8个分子表达相对高为特点,而聚类2以8个分子表达相对低为特点,且分别与遗传因素有关,聚类1中家系冠心病血瘀证：非家系冠心病血瘀证为14/4,聚类2中家系冠心病血瘀证：非家系冠心病血瘀证为10/17,二者间具有统计学差异。
4.通过基因表达谱芯片技术分析的6组具有较好的区分度,通过对挑选出的5个差异基因进行实时荧光RT-PCR验证,与芯片结果有较好的一致性,芯片研究结果的可信度较高。
5.对家系和非家系冠心病血瘀证差异表达基因谱进行比较,结果表明若以FC>2或FC<0.5的标准筛选,二者共同具有的基因探针数78个,分别只占家系和非家系冠心病血瘀证基因表达谱的比例为4.12%(78/1893)和3.69%(78/2113)；以FC>3或FC<1/3筛选,二者共同具有的基因探针数35个,分别只占家系和非家系冠心病血瘀证基因表达谱的比例为5.11%(35/685)和4.62%(35/758)。且二者的不同表达基因谱在GO功能分布和Pathway通路中存在明显的区别。
两组人群的异质性表达基因探针中有173个差异探针是相同的,只是表达的方向存在差异,即一组是“up”则另一组是‘'down"。对这部分基因通过SAS系统在线注释,结果表明目前这部分探针中已有81个基因在数据库中有注释。进一步对基因本体(GO)分析和通路(pathway)进行分析,以Enrichment test p value<0.05为筛选标准,通过筛选GO分析共有归属于不同类的34个有价值性的基因,Pathway分析可获得43个有意义的通路。在34个GO功能注释的基因中可能与冠心病血瘀证有关的基因有CEACAM1、CXCL1和IL8。而从43个通路来看,Adhesion and Diapedesis of Lymphocytes、Cells and Molecules involved in local acute inflammatory response、Adhesion Molecules on Lymphocyte、Free Radical Induced Apoptosis、Eph Kinasesand ephrins support platelet aggregation.Monocyte and its Surface Molecules. B Cell Survival Pathway. CD40L Signaling Pathway. Adhesion and Diapedesis of Granulocytes. IL17Signaling Pathway. Ras-Independent pathway in NK cell-mediated cytotoxicity. PTEN dependent cell cycle arrest and apoptosis.Cytokine Network和Cytokines and Inflammatory Response等炎症免疫相关通路也可能与冠心病血瘀证有潜在的关联。然而在家系冠心病血瘀证和非家系冠心病血瘀证两组中,这些基因或通路构成基因的表达是相反的。
6.通过异病同证、同病异证相结合的方式筛选冠心病血瘀证的易感表达基因。其中异病同证方法筛选出易感基因探针数739个(FC>2或FC<0.5,已注释基因406个)或者易感基因探针数162个(FC>3或FC<1/3,已有注释的基因93个)；同病异证方法筛选出易感基因探针数1357个(FC>2或FC<0.5,已有注释的基因830个)或易感基因探针数516个(FC>3或FC<1/3,已有注释的基因316个)。
7.从易感基因中选取2条基因,即低密度脂蛋白受体相关蛋白12(LRP12)和Kruppel样因子5(KLF5),在23例人群(冠心病血瘀证组14例,健康人组9例)中进行启动子甲基化状态的分析。结果表明冠心病血瘀证与健康人之间,两基因的甲基化状态无差异；从冠心病血瘀证组内来看,不同个体间此二者基因的启动子甲基化状态存在差别的,分别存在甲基化、不完全甲基化和非甲基化三种状态。
[结论]
1.血府逐瘀汤治疗冠心病血瘀证的疗效的差异和冠心病血瘀证客观化研究结果的差异,提示冠心病血瘀证可能存在着证本质的异质性。
2.冠心病血瘀证的表征存在着差异,这种差异可能与遗传因素有关,提示冠心病血瘀证的证本质可能存在遗传异质性。
3.冠心病血瘀证的家系和非家系患者的基因表达谱结果的差异表明,冠心病血瘀证的证本质可能存在遗传异质性。
4.冠心病血瘀证易感基因的启动子甲基化结果的差异结局提示,冠心病血瘀证的证本质异质性与表观遗传学的异质性可能有关。
总之,从课题的研究结果来看,冠心病血瘀证的证本质存在着异质性,这种异质性产生的原因可能涉及到遗传和表观遗传等一系列调控网络体系。但这种证本质异质性也可能从表征的差别中有所体现。
Objective:By analyzing about the characterization of coronary heart disease with blood stasis syndrome, which including the symptoms, signs and prethrombotic state moleculars, gene expression profiling and susceptible gene promoter methylation status, to discuss that if coronary heart disease with blood stasis syndrome essence exists heterogeneity?
Method:It is the literature research firstly. By searching the CNKI database, the VIP database, the Wanfang database, the Chinese biomedical literature database and the Pubmed database, to query the clinical literature on blood stasis syndrome of coronary heart disease roundly. The clinical literatures mainly include two aspects. One is the studies of the clinical effect of Xuefu Zhuyu decoction in the treatment of coronary heart disease with blood stasis. Other is objective studies of the coronary heart disease with blood stasis syndrome, including using high-throughput techniques (genome, proteome, metabolome, etc) and other microscopic indexes. Then we extract the information about the documents, and carry the system evaluation by utilizing the meta analysis method. Secondly, to analyze the clinical characterization of the coronary heart disease with blood stasis, including symptoms, signs and prethrombotic state molecule, by using the systematic cluster analysis method and according to the principle with large spacing between groups, small spacing within a group. Thirdly, micro array technology was used to analyze gene expression about study groups (family groups:coronary heart disease with blood stasis syndrome, non blood stasis syndrome, non blood stasis coronary heart disease and healthy people; non family group: health people, blood stasis in patients with coronary heart disease). And real time fluorescent quantitative RT-PCR technique was used on numerous differences selected gene expression validation. More, by methylation specific PCR (MS-PCR) was applied to analyze the methylation status of the promoter.
Results:The inclusion of the nearly ten years (2003-2012),18articles about Xuefu Zhuyu Decoction in the treatment of angina pectoris of coronary heart disease with blood stasis syndrome were analyzed, and the results showed that Xuefu Zhuyu Decoction could improve the performance of the TCM syndrome curative effect, angina pectoris effects and ECG efficacy, but the independent research within the effect extent existed the heterogeneity of cured, effective, ineffective and worse. Study on coronary heart disease of blood stasis with high-throughput technology access total21literatures study, included the gene expression profile of3articles,11articles on proteomics,6Metabonomics studies. And studies on other microscopic indexes have35articles(13single nucleotide polymorphism studies of peripheral blood white cells,22other articles).some studies, which met the inclusion criteria about ACE, ApoE and F VII gene polymorphism research, other microscopic indicators (nitric oxide, von Willebrand factor, endothelin, soluble intercellular adhesion molecule, tissue plasminogen activator inhibitor-1, tissue plasminogen activator) was analyzed by meta. The results showed that whether the research analysis method or not, the existing research have different degrees of heterogeneity in outcome indicators. In66patients with the coronary heart disease with blood stasis (including39family cases of patients with coronary heart disease with blood stasis,27non family cases of coronary heart disease with blood stasis), system cluster analysis of symptoms and signs in10diagnostic elements of tightness in the chest, the blood stasis prickling, face purple, lip green, nail green, tongue color, tongue petechiae, Knotted and Intermittent Pulse.The results showed that the clinical characterization of blood stasis syndrome could be divided into two subgroups. Cluster1feature:the combinations of symptoms (tightness in the chest, lip green and face purple) as the main performance, other symptoms and signs were rare. Cluster2features:the combinations of symptoms (tightness in the chest, lip green, face purple, blood stasis prickling and the tongue color) were the most common symptoms, tongue petechiaes was extremely atypical symptoms. Through the statistical analysis of the2subgroups respectively with the family blood stasis syndrome of coronary heart disease, which has a genetic background and non-family blood stasis syndrome of coronary heart disease without the chromosomal background. In cluster1, family patients with blood stasis syndrome had12cases, but non family patients with blood stasis syndrome had21cases. However, in cluster2, family patients had27cases, but non family previously had6cases, with statistical significance. In patients with coronary heart disease with blood stasis of the prethrombotic state molecular (PTS) included the8markers: the levels of plasma thrombomodulin (TM), P-selectin, prothrombin fragment1,2(F1+2), soluble fibrin monomer complex(SFMC), tissue type plasminogen activator (tPA), plasminogen activator inhibitor (PAI-1), thrombin antithrombin(TAT) and C protein peptide (PCP) as the object of system clustering analysis and statistics. The results show that the family of blood stasis syndrome of coronary heart disease and family health had statistical differences in TM, P-Selectin,F1+2, t-PA, TAT, PCP six molecules, there was no significant difference in non family blood stasis syndrome and non-family health of human, and the family of blood stasis syndrome of coronary heart disease and non-family of blood stasis syndrome of coronary heart disease, In t-PA, PAI-1, TAT three molecules there was statistical difference.Hierarchical cluster analysis (Ward) showed that PTS molecules could be clustered into two groups. In cluster1,8molecule expressed higher as the characteristic, and in cluster2,8molecules expressed relatively low as the characteristics, and were associated with hereditary factors. In cluster1,14pedigrees of blood stasis syndrome of coronary heart disease compared with4non family cases of coronary heart disease. In cluster2, family blood stasis syndrome of coronary heart disease had10cases, but non family Blood Stasis Syndrome of coronary heart disease had17cases, with statistical difference.6groups had a better distinction in gene expression by microarray analysis.5selected genes were detected by real-time RT-PCR validation and were better consistent with the chip results. So the chip results were reliable. The pedigree of blood stasis syndrome compared with non family in gene expression spectrum, results showed that by screening with FC<2or FC<0.5standards, the two groups had the same78gene probe number, respectively, accounted for only family and non family blood stasis syndrome gene expression ratio was4.12%(78/1893) and3.69%(78/2113). With FC>3or FC<1/3screening, the two groups had the same35gene probe number, respectively, accounted for single family and non family blood stasis syndrome gene expression ratio was5.11%(35/685) and4.62%(35/758).And the two distinct gene expression spectrum exist an obvious difference in the GO distribution and the pathway. Two groups of heterogeneous expression gene probe in173different probe are the same, but there is a difference in the expression of the direction, i.e. a group of "up" but another group of "down". This part of the gene by SAS system on-line comments, results show that at present this part probe had81genes annotated in the database. The gene ontology (GO) analysis and path analysis (pathway), Enrichment test p value<0.05as the selection criteria, by screening for GO analysis of a total of34valuable genes belong to different classes, Pathway analysis can obtain43meaningful pathway.In34GO functional annotations of genes may be associated with blood stasis syndrome gene were CEACAM1, CXCL1and IL8.From the43pathways, Adhesion and Diapedesis of Lymphocytes, Cells and Molecules involved in local acute inflammatory response, Adhesion Molecules on Lymphocyte, Free Radical Induced Apoptosis, Eph Kinases and ephrins support platelet aggregation, Monocyte and its Surface Molecules, B Cell Survival Pathway, CD40L Signaling Pathway, Adhesion and Diapedesis of Granulocytes, IL17Signaling Pathway, Ras-Independent pathway in NK cell-mediated cytotoxicity, PTEN dependent cell cycle arrest and apoptosis, Cytokine Network and Cytokines and Inflammatory Response and other inflammatory immune related pathways may also had potential association with blood stasis syndrome of coronary heart disease. However, in the family blood stasis syndrome and non-family blood stasis syndrome of coronary heart disease in two groups, these genes or pathways of gene expression were the opposite. Expression of susceptibility genes were screened through the identical syndrome in different diseases, the same disease with different combination way.The same syndrome in different diseases screening method of susceptible gene probe number739(FC<2or FC<0.5annotated genes,406) or susceptible gene probe number162(FC<3or FC<1/3,93genes have been annotated); the same disease with different method of screening susceptibility genes probe number1357(FC<2or FC<0.5, has annotated gene830) or susceptible gene probe number516(FC<3or FC <1/3,316genes have been annotated).2genes, namely low density lipoprotein receptor related protein12(LRP12) and Kruppel like factor5(KLF5), were selected from the susceptible genes. In23cases of the crowd (14cases of blood stasis syndrome group, and9cases of healthy adults) were analyzed the methylation status of the selected genes.The results show that between blood stasis syndrome and healthy people, no difference in methylation status of two genes. From the point of view of coronary heart disease with blood stasis group, promoter methylation status between different individuals of these two genes exist difference, there were methylated, not fully methylated and non-methylated three states.
Conclusion:1. The difference of curative effect of Xuefu Zhuyu Decoction in treating coronary heart disease with blood stasis and the objective results of blood stasis syndrome in the research, suggested the heterogeneity of syndrome essence.2. There exist differences in characterization of coronary heart disease with blood stasis. The differences may be related to hereditary factors, prompted that coronary heart disease with blood stasis syndrome essence may be genetic heterogeneity.3. Blood stasis syndrome pedigrees patients with non home show differences gene expression spectrum results, coronary heart disease with blood stasis syndrome essence may be genetic heterogeneity.4. Differences in outcome suggest that blood stasis syndrome in coronary heart disease susceptibility gene promoter methylation results, the nature of heterogeneity and scale heterogeneity in genetics might be related to coronary heart disease with blood stasis syndrome. In conclusion, from the results of view, coronary heart disease with blood stasis syndrome essence exists heterogeneity. This heterogeneity generated may be related to genetic and epigenetic and a series of control network system.However, the nature of heterogeneity may also reflect from the characterization of the differences.
通过对冠心病血瘀证的表征(包括症状体征与血栓前状态分子)、基因表达谱和易感基因启动子甲基化状态的研究,探讨冠心病血瘀证的证本质是否存在着异质性特征。
[方法]
1.文献研究：通过检索中国知网(CNKI)、维普(CQVIP)、万方(WANFANG DATA)、中国生物医学文献数据库(CBM)及Pubmed等数据库,查询有关冠心病血瘀证的临床文献,主要集中在两个方面：①血府逐瘀汤治疗冠心病血瘀证的临床疗效；②冠心病血瘀证的客观化研究,包含采用高通量技术(基因组、蛋白质组、代谢组)及其他微观指标的文献。提取纳入文献的有关信息,采用meta分析等方法进行系统评价
2.临床研究：采用系统聚类分析等方法,以组间间距大、组内间距小的原则,对冠心病血瘀证患者的临床表征---症状体征与血栓前状态分子进行分析。
3.实验研究：应用基因表达谱芯片技术对研究各组(家系组：冠心病血瘀证、冠心病非血瘀证、非冠心病血瘀证、健康人；非家系组：冠心病血瘀证、健康人)的基因表达情况进行分析；采用实时荧光定量RT-PCR技术对挑选的若干差异表达基因进行验证；采用甲基化特异性PCR技术(MS-PCR)对若干基因启动子的甲基化状态进行分析。
[结果]
1.对纳入的近十年(2003-2012年)有关血府逐瘀汤治疗冠心病心绞痛血瘀证的18篇文献进行分析,结果表明血府逐瘀汤在改善中医证候疗效、心绞痛疗效和心电图疗效方面均有较好表现,但各独立研究内部在疗效程度上存在痊愈、有效、无效和加重的异质性。
2.各数据库获取的冠心病血瘀证高通量研究的文献共21篇(基因表达谱研究3篇,蛋白质谱研究11篇,代谢组学研究6篇),其他微观指标的研究35篇(外周血白细胞DNA单核苷酸多态性研究13篇,其他22篇)。其中对符合纳入标准的ACE、ApoE和FⅦ基因多态性研究、其他微观指标研究(一氧化氮、血管性血友病因子、内皮素、可溶性细胞间黏附分子、组织纤维蛋白溶解酶原激活物抑制剂-1、组织型纤溶酶原激活物)进行了meta分析,结果表明不管是高通量技术研究还是还原分析方法的研究,现有的各研究在结局指标间均存在不同程度的异质性。
3.对纳入的冠心病血瘀证患者66例(其中家系冠心病血瘀证患者39例,非家系冠心病血瘀证患者27例),以血瘀证的10类诊断要素胸闷、针刺痛、面紫、唇青、甲青、舌色、舌瘀斑、脉涩、脉结、脉代的评分结果进行症状体征的系统聚类分析(Ward法),结果表明,冠心病血瘀证的临床表征可以分为两个亚类,聚类1特征：以(胸闷+唇青+面紫)的症状组合为主要表现,其他症状体征少见；聚类2特征：以(胸闷+唇青+面紫)+(刺痛+舌色)的症状组合为常见症状,且舌瘀斑为极少见症状。通过统计学分析这2亚类分别与具有遗传背景的家系冠心病血瘀证和无遗传背景的非家系冠心病血瘀证有相关性,聚类1中家系冠心病血瘀证患者：非家系冠心病血瘀证患者为12/21(人),聚类2中非家系冠心病血瘀证患者：家系冠心病血瘀证患者为27/6(人),具有统计学意义。
以冠心病血瘀证患者的外周血血栓前状态分子(PTS)的8个标记物：血浆血栓调节蛋白(TM)、血小板表面P-选择素(P-Selectin)、凝血酶原片段1、2(F1+2)、可溶性纤维蛋白单体(SFMC)、组织型纤溶酶原激活物(tPA)、纤溶酶原激活物抑制物(PAI-1)、凝血酶-抗凝血酶Ⅲ复合物(TAT)和蛋白质C肽(PCP)为对象进行系统聚类和统计学分析。结果表明家系冠心病血瘀证与家系健康人比较在TN、P-Selectiin、F1+2、t-PA、TAT、PCP六个分子上有统计学差异,非家系冠心病血瘀证和非家系健康人间无统计学差异,而家系冠心病血瘀证与非家系冠心病血瘀证间在t-PA、PAI-1、TAT三个分子上有统计学差异。系统聚类分析(Ward法)表明PTS分子可以聚为两类,聚类1以8个分子表达相对高为特点,而聚类2以8个分子表达相对低为特点,且分别与遗传因素有关,聚类1中家系冠心病血瘀证：非家系冠心病血瘀证为14/4,聚类2中家系冠心病血瘀证：非家系冠心病血瘀证为10/17,二者间具有统计学差异。
4.通过基因表达谱芯片技术分析的6组具有较好的区分度,通过对挑选出的5个差异基因进行实时荧光RT-PCR验证,与芯片结果有较好的一致性,芯片研究结果的可信度较高。
5.对家系和非家系冠心病血瘀证差异表达基因谱进行比较,结果表明若以FC>2或FC<0.5的标准筛选,二者共同具有的基因探针数78个,分别只占家系和非家系冠心病血瘀证基因表达谱的比例为4.12%(78/1893)和3.69%(78/2113)；以FC>3或FC<1/3筛选,二者共同具有的基因探针数35个,分别只占家系和非家系冠心病血瘀证基因表达谱的比例为5.11%(35/685)和4.62%(35/758)。且二者的不同表达基因谱在GO功能分布和Pathway通路中存在明显的区别。
两组人群的异质性表达基因探针中有173个差异探针是相同的,只是表达的方向存在差异,即一组是“up”则另一组是‘'down"。对这部分基因通过SAS系统在线注释,结果表明目前这部分探针中已有81个基因在数据库中有注释。进一步对基因本体(GO)分析和通路(pathway)进行分析,以Enrichment test p value<0.05为筛选标准,通过筛选GO分析共有归属于不同类的34个有价值性的基因,Pathway分析可获得43个有意义的通路。在34个GO功能注释的基因中可能与冠心病血瘀证有关的基因有CEACAM1、CXCL1和IL8。而从43个通路来看,Adhesion and Diapedesis of Lymphocytes、Cells and Molecules involved in local acute inflammatory response、Adhesion Molecules on Lymphocyte、Free Radical Induced Apoptosis、Eph Kinasesand ephrins support platelet aggregation.Monocyte and its Surface Molecules. B Cell Survival Pathway. CD40L Signaling Pathway. Adhesion and Diapedesis of Granulocytes. IL17Signaling Pathway. Ras-Independent pathway in NK cell-mediated cytotoxicity. PTEN dependent cell cycle arrest and apoptosis.Cytokine Network和Cytokines and Inflammatory Response等炎症免疫相关通路也可能与冠心病血瘀证有潜在的关联。然而在家系冠心病血瘀证和非家系冠心病血瘀证两组中,这些基因或通路构成基因的表达是相反的。
6.通过异病同证、同病异证相结合的方式筛选冠心病血瘀证的易感表达基因。其中异病同证方法筛选出易感基因探针数739个(FC>2或FC<0.5,已注释基因406个)或者易感基因探针数162个(FC>3或FC<1/3,已有注释的基因93个)；同病异证方法筛选出易感基因探针数1357个(FC>2或FC<0.5,已有注释的基因830个)或易感基因探针数516个(FC>3或FC<1/3,已有注释的基因316个)。
7.从易感基因中选取2条基因,即低密度脂蛋白受体相关蛋白12(LRP12)和Kruppel样因子5(KLF5),在23例人群(冠心病血瘀证组14例,健康人组9例)中进行启动子甲基化状态的分析。结果表明冠心病血瘀证与健康人之间,两基因的甲基化状态无差异；从冠心病血瘀证组内来看,不同个体间此二者基因的启动子甲基化状态存在差别的,分别存在甲基化、不完全甲基化和非甲基化三种状态。
[结论]
1.血府逐瘀汤治疗冠心病血瘀证的疗效的差异和冠心病血瘀证客观化研究结果的差异,提示冠心病血瘀证可能存在着证本质的异质性。
2.冠心病血瘀证的表征存在着差异,这种差异可能与遗传因素有关,提示冠心病血瘀证的证本质可能存在遗传异质性。
3.冠心病血瘀证的家系和非家系患者的基因表达谱结果的差异表明,冠心病血瘀证的证本质可能存在遗传异质性。
4.冠心病血瘀证易感基因的启动子甲基化结果的差异结局提示,冠心病血瘀证的证本质异质性与表观遗传学的异质性可能有关。
总之,从课题的研究结果来看,冠心病血瘀证的证本质存在着异质性,这种异质性产生的原因可能涉及到遗传和表观遗传等一系列调控网络体系。但这种证本质异质性也可能从表征的差别中有所体现。
Objective:By analyzing about the characterization of coronary heart disease with blood stasis syndrome, which including the symptoms, signs and prethrombotic state moleculars, gene expression profiling and susceptible gene promoter methylation status, to discuss that if coronary heart disease with blood stasis syndrome essence exists heterogeneity?
Method:It is the literature research firstly. By searching the CNKI database, the VIP database, the Wanfang database, the Chinese biomedical literature database and the Pubmed database, to query the clinical literature on blood stasis syndrome of coronary heart disease roundly. The clinical literatures mainly include two aspects. One is the studies of the clinical effect of Xuefu Zhuyu decoction in the treatment of coronary heart disease with blood stasis. Other is objective studies of the coronary heart disease with blood stasis syndrome, including using high-throughput techniques (genome, proteome, metabolome, etc) and other microscopic indexes. Then we extract the information about the documents, and carry the system evaluation by utilizing the meta analysis method. Secondly, to analyze the clinical characterization of the coronary heart disease with blood stasis, including symptoms, signs and prethrombotic state molecule, by using the systematic cluster analysis method and according to the principle with large spacing between groups, small spacing within a group. Thirdly, micro array technology was used to analyze gene expression about study groups (family groups:coronary heart disease with blood stasis syndrome, non blood stasis syndrome, non blood stasis coronary heart disease and healthy people; non family group: health people, blood stasis in patients with coronary heart disease). And real time fluorescent quantitative RT-PCR technique was used on numerous differences selected gene expression validation. More, by methylation specific PCR (MS-PCR) was applied to analyze the methylation status of the promoter.
Results:The inclusion of the nearly ten years (2003-2012),18articles about Xuefu Zhuyu Decoction in the treatment of angina pectoris of coronary heart disease with blood stasis syndrome were analyzed, and the results showed that Xuefu Zhuyu Decoction could improve the performance of the TCM syndrome curative effect, angina pectoris effects and ECG efficacy, but the independent research within the effect extent existed the heterogeneity of cured, effective, ineffective and worse. Study on coronary heart disease of blood stasis with high-throughput technology access total21literatures study, included the gene expression profile of3articles,11articles on proteomics,6Metabonomics studies. And studies on other microscopic indexes have35articles(13single nucleotide polymorphism studies of peripheral blood white cells,22other articles).some studies, which met the inclusion criteria about ACE, ApoE and F VII gene polymorphism research, other microscopic indicators (nitric oxide, von Willebrand factor, endothelin, soluble intercellular adhesion molecule, tissue plasminogen activator inhibitor-1, tissue plasminogen activator) was analyzed by meta. The results showed that whether the research analysis method or not, the existing research have different degrees of heterogeneity in outcome indicators. In66patients with the coronary heart disease with blood stasis (including39family cases of patients with coronary heart disease with blood stasis,27non family cases of coronary heart disease with blood stasis), system cluster analysis of symptoms and signs in10diagnostic elements of tightness in the chest, the blood stasis prickling, face purple, lip green, nail green, tongue color, tongue petechiae, Knotted and Intermittent Pulse.The results showed that the clinical characterization of blood stasis syndrome could be divided into two subgroups. Cluster1feature:the combinations of symptoms (tightness in the chest, lip green and face purple) as the main performance, other symptoms and signs were rare. Cluster2features:the combinations of symptoms (tightness in the chest, lip green, face purple, blood stasis prickling and the tongue color) were the most common symptoms, tongue petechiaes was extremely atypical symptoms. Through the statistical analysis of the2subgroups respectively with the family blood stasis syndrome of coronary heart disease, which has a genetic background and non-family blood stasis syndrome of coronary heart disease without the chromosomal background. In cluster1, family patients with blood stasis syndrome had12cases, but non family patients with blood stasis syndrome had21cases. However, in cluster2, family patients had27cases, but non family previously had6cases, with statistical significance. In patients with coronary heart disease with blood stasis of the prethrombotic state molecular (PTS) included the8markers: the levels of plasma thrombomodulin (TM), P-selectin, prothrombin fragment1,2(F1+2), soluble fibrin monomer complex(SFMC), tissue type plasminogen activator (tPA), plasminogen activator inhibitor (PAI-1), thrombin antithrombin(TAT) and C protein peptide (PCP) as the object of system clustering analysis and statistics. The results show that the family of blood stasis syndrome of coronary heart disease and family health had statistical differences in TM, P-Selectin,F1+2, t-PA, TAT, PCP six molecules, there was no significant difference in non family blood stasis syndrome and non-family health of human, and the family of blood stasis syndrome of coronary heart disease and non-family of blood stasis syndrome of coronary heart disease, In t-PA, PAI-1, TAT three molecules there was statistical difference.Hierarchical cluster analysis (Ward) showed that PTS molecules could be clustered into two groups. In cluster1,8molecule expressed higher as the characteristic, and in cluster2,8molecules expressed relatively low as the characteristics, and were associated with hereditary factors. In cluster1,14pedigrees of blood stasis syndrome of coronary heart disease compared with4non family cases of coronary heart disease. In cluster2, family blood stasis syndrome of coronary heart disease had10cases, but non family Blood Stasis Syndrome of coronary heart disease had17cases, with statistical difference.6groups had a better distinction in gene expression by microarray analysis.5selected genes were detected by real-time RT-PCR validation and were better consistent with the chip results. So the chip results were reliable. The pedigree of blood stasis syndrome compared with non family in gene expression spectrum, results showed that by screening with FC<2or FC<0.5standards, the two groups had the same78gene probe number, respectively, accounted for only family and non family blood stasis syndrome gene expression ratio was4.12%(78/1893) and3.69%(78/2113). With FC>3or FC<1/3screening, the two groups had the same35gene probe number, respectively, accounted for single family and non family blood stasis syndrome gene expression ratio was5.11%(35/685) and4.62%(35/758).And the two distinct gene expression spectrum exist an obvious difference in the GO distribution and the pathway. Two groups of heterogeneous expression gene probe in173different probe are the same, but there is a difference in the expression of the direction, i.e. a group of "up" but another group of "down". This part of the gene by SAS system on-line comments, results show that at present this part probe had81genes annotated in the database. The gene ontology (GO) analysis and path analysis (pathway), Enrichment test p value<0.05as the selection criteria, by screening for GO analysis of a total of34valuable genes belong to different classes, Pathway analysis can obtain43meaningful pathway.In34GO functional annotations of genes may be associated with blood stasis syndrome gene were CEACAM1, CXCL1and IL8.From the43pathways, Adhesion and Diapedesis of Lymphocytes, Cells and Molecules involved in local acute inflammatory response, Adhesion Molecules on Lymphocyte, Free Radical Induced Apoptosis, Eph Kinases and ephrins support platelet aggregation, Monocyte and its Surface Molecules, B Cell Survival Pathway, CD40L Signaling Pathway, Adhesion and Diapedesis of Granulocytes, IL17Signaling Pathway, Ras-Independent pathway in NK cell-mediated cytotoxicity, PTEN dependent cell cycle arrest and apoptosis, Cytokine Network and Cytokines and Inflammatory Response and other inflammatory immune related pathways may also had potential association with blood stasis syndrome of coronary heart disease. However, in the family blood stasis syndrome and non-family blood stasis syndrome of coronary heart disease in two groups, these genes or pathways of gene expression were the opposite. Expression of susceptibility genes were screened through the identical syndrome in different diseases, the same disease with different combination way.The same syndrome in different diseases screening method of susceptible gene probe number739(FC<2or FC<0.5annotated genes,406) or susceptible gene probe number162(FC<3or FC<1/3,93genes have been annotated); the same disease with different method of screening susceptibility genes probe number1357(FC<2or FC<0.5, has annotated gene830) or susceptible gene probe number516(FC<3or FC <1/3,316genes have been annotated).2genes, namely low density lipoprotein receptor related protein12(LRP12) and Kruppel like factor5(KLF5), were selected from the susceptible genes. In23cases of the crowd (14cases of blood stasis syndrome group, and9cases of healthy adults) were analyzed the methylation status of the selected genes.The results show that between blood stasis syndrome and healthy people, no difference in methylation status of two genes. From the point of view of coronary heart disease with blood stasis group, promoter methylation status between different individuals of these two genes exist difference, there were methylated, not fully methylated and non-methylated three states.
Conclusion:1. The difference of curative effect of Xuefu Zhuyu Decoction in treating coronary heart disease with blood stasis and the objective results of blood stasis syndrome in the research, suggested the heterogeneity of syndrome essence.2. There exist differences in characterization of coronary heart disease with blood stasis. The differences may be related to hereditary factors, prompted that coronary heart disease with blood stasis syndrome essence may be genetic heterogeneity.3. Blood stasis syndrome pedigrees patients with non home show differences gene expression spectrum results, coronary heart disease with blood stasis syndrome essence may be genetic heterogeneity.4. Differences in outcome suggest that blood stasis syndrome in coronary heart disease susceptibility gene promoter methylation results, the nature of heterogeneity and scale heterogeneity in genetics might be related to coronary heart disease with blood stasis syndrome. In conclusion, from the results of view, coronary heart disease with blood stasis syndrome essence exists heterogeneity. This heterogeneity generated may be related to genetic and epigenetic and a series of control network system.However, the nature of heterogeneity may also reflect from the characterization of the differences.
引文
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