【作者】 何文娟；

【导师】 胡豫；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 第一部分冠心病血栓形成相关生化标志物研究目的凝血、抗凝、纤溶及内皮功能异常与冠心病的发生、发展密切相关,已有研究发现,在心肌梗死发生之前可以检测到患者体内的高凝状态。单个研究较少涉及对多个血栓相关指标的探讨,因此,本研究的目的是评估多个血栓形成相关因子及一些其它危险因素对冠心病的影响。方法冠心病组共56例,包括26例稳定型心绞痛(stable angina pectoris, SAP)及30例急性冠脉综合征(acute coronary syndrome, ACS)患者,均为2006年1月至7月在武汉协和医院心内科住院的患者;对照组54例,均为同期在该院其它科室住院的患者,排除了血栓栓塞性疾病、糖尿病、高血压病、恶性肿瘤、急慢性肝肾疾病及结缔组织病。病例组和对照组在年龄、性别及样本采集时间上均匹配。蛋白C(protein C, PC)、游离蛋白S(free protein S, FPS)、总蛋白S(total protein S, TPS)、凝血酶调节蛋白(thrombomodulin, TM)、活化凝血因子VII(activated factor VII, FVIIa)、凝血因子VII抗原(factor VII Antigen, FVIIag)、P选择素、组织型纤溶酶原激活剂(tissue-type plasminogen activator, tPA)及纤溶酶原激活剂抑制物-1(plasminogen activator inhibitor-1, PAI-1)的血浆水平均采用酶联免疫吸附法测定,组织因子活性(tissue factor activity, TFc)采用发色底物法测定,活化蛋白C(activated protein C, APC)比率、凝血酶原时间(prothrombin time, PT)、活化部分凝血活酶时间(activated partial thromboplastin time, APTT)、纤维蛋白原(fibrinogen, Fbg)、D二聚体(D-dimmer, DD)及凝血酶时间(thrombin time, TT)采用全自动血凝分析仪(Sysmex CA-7000,日本)检测。血脂水平由协和医院检验科统一测定(全自动生化分析仪7170A,Hitachi,日本)。结果ACS组中TFc高于SAP组(P <0.05)和对照组(P <0.01);ACS组中FVIIag水平明显高于对照组(P<0.05);ACS组和SAP组中的FVIIa(P<0.01)、TM(P<0.05)及APC比率<2.4的阳性率(P<0.01)均明显高于对照组,APC比率均明显低于对照组(P<0.01);蛋白C、游离蛋白S、总蛋白S、P选择素、tPA、PAI-1血浆水平及APC抵抗阳性率在各组间均无统计学差异;Logistic回归分析显示,FVIIa及TFc的水平增高是冠心病的危险因素,高密度脂蛋白和APC比率增高是冠心病的保护性因素。结论SAP和ACS患者均可出现外源性凝血途径的激活、对APC反应性的下降以及内皮功能的受损或活化;血浆中TFc增强可能预示了急性冠脉事件的发生;血浆FVIIa及TFc与冠心病的发病明显相关。第二部分冠心病血栓形成相关蛋白标志物研究目的利用表面增强激光解析/电离飞行时间质谱仪(SELDI-TOF-MS)及弱阳离子交换蛋白质芯片CM10检测急性心肌梗死患者和对照者血浆中蛋白质指纹图谱,筛选出不同组之间的差异蛋白质标志物并建立相应的诊断模型。方法急性心肌梗死组77例,均为2006年1月～7月在武汉协和医院心内科住院患者,急性脑梗死组32例,为同期在该院神经内科住院患者,正常对照组60例。病例组和对照组在年龄、性别及样本采集时间上均匹配。将受试者血浆与CM10蛋白质芯片结合后,置入SELDI-TOF-MS系统进行蛋白质质谱测定,采用浙江大学肿瘤中心蛋白芯片数据分析系统对数据进行分析,找出差异蛋白质并建立相应诊断模型。结果用SELDI-TOF-MS技术筛选出由5个有显著差异的蛋白质峰(质荷比分别为3321.6、16099.3、8058.7、15933.0和16286.7)组合构建的诊断模型,区分急性心肌梗死患者和正常人的诊断特异性和灵敏度分别为95%和88.3%;筛选出由17个蛋白质峰(质荷比分别为6682.6、6485.6、3321.6、16682.9、16297.7、7977.5、3424.7、4821.1、11748.7、4775.3、8977.3、9094.8、4361.4、11479.9、2941.1、11399.7和2462.3)组合构建的诊断模型,区分急性心肌梗死者和急性脑梗死者的诊断特异性和灵敏度均为100%。结论SELDI-TOF-MS技术在急性心肌梗死的诊断上具有较高灵敏度和特异性,发现的蛋白质峰可能对急性心肌梗死的早期诊断具有一定价值。第三部分MASA技术检测冠心病血栓形成相关基因变化目的冠心病是一种多因素疾病,遗传因素也参与其发生和发展。本研究拟采用液相基因芯片技术(MASA)检测急性心肌梗死患者的7种基因多态性,并对阳性结果进行验证,探讨MASA技术检测基因变化的有效性。方法一共有34例急性心肌梗死患者纳入研究,均为2006年1月～7月在武汉协和医院心内科住院患者。从血液中提取患者DNA并经PCR扩增,把针对7种基因改变(包括FV Leiden G/A突变、Fbg Beta -455 G/A突变、Fbg Beta -148 C/T突变、Fbg Beta 448 G/A突变、FVII -323 10bp插入突变、凝血酶原20210 G/A突变、MTHFR 677 C/T突变)的核酸探针以共价方式结合到特定荧光编码的微球上,把针对不同检测物的微球混合,加入PCR扩增产物杂交并标记荧光物质,利用Luminex 100?多功能流式点阵仪进行检测,采用Sanger双脱氧链中止法对阳性结果进行测序验证。结果34例患者的MASA检测结果示,有19例存在基因变化,包括FV Leiden突变4例,MTHFR 677 C/T突变14例,纤维蛋白原β链448 G/A突变(Fbgβ448 G/A)5例,其中2例FV Leiden分别合并Fbgβ448 G/A突变和MTHFR677 C/T突变,2例MTHFR 677 C/T突变合并Fbgβ448 G/A突变。阳性结果经测序之后,证实一共15例患者存在基因变化,FV Leiden仅有1例突变,MTHFR 677 C/T突变12例,Fbgβ448 G/A突变4例,其中1例FV Leiden合并Fbgβ448 G/A突变,1例MTHFR 677 C/T突变合并Fbgβ448 G/A突变,MASA检测的阳性病例的总体正确率为73.9%。结论液态基因芯片检测急性心肌梗死基因改变具有可行性,在基因研究中尚需对MASA技术进行进一步改进。更多还原

【Abstract】 Part I Study on Biochemical Markers of Thrombosis in Coronary Heart DiseaseObjective There are close relationships between disfunctions of coagulation, anticoagulation, fibrinolysis as well as endothelium and development of coronary heart disease. Some studies found that during pre-infarction phases, a hypercoagulable state may be detectable. Single research referring to multiple thrombosis factors is rare. Thus, the main objective of this study was to evaluate the effects of multiple thrombosis factors as well as some risk factors on coronary heart disease. Methods 56 patients with coronary artery disease (including 26 stable angina pectoris and 30 acute coronary syndromes) admitted to department of cardiology in Wuhan Union Hospital and 54 control patients excluded thromboembolic disease, diabetes mellitus, hypertension, malignant tumor, acute/chronic liver or kidney disease and connective tissue disease were enrolled from January to July 2006. Coronary artery disease group and control group were matched for age, sex and sampling time. Plasma levels of protein C, free protein S, total protein S, thrombomodulin, activated factor VII, factor VII Antigen, P-selectin, tissue-type plasminogen activator, plasminogen activator inhibitor-1 were measured by enzyme linked immunosorbent assay, activity of tissue factor was measured by chromogenic activity assay, and activated protein C ratio, prothrombin time, activated partial thromboplastin time, fibrinogen, D-dimmer and thrombin time were detected by full-automated coagulation analyzer (Sysmex CA-7000, Japan). Levels of blood lipids were assayed by Department of Laboratory of Union Hospital. Results The levels of tissue factor activity in patients with acute coronary syndromes were found to be significantly higher than those in controls (p<0.01) or in stable angina patients (p<0.05); the levels of FVIIag in ACS patients were significantly higher than those of controls; compared with controls, the plasma levels of FVIIa (P<0.01) and TM (P<0.05) as well as prevalences of APC ratio below 2.4 (P<0.01) in ACS and SAP groups were higher, however, APC ratios were lower (P<0.01). The result of binary logistic regression analysis showed that activated factor VII (OR 2.680, 95%CI 1.539-4.665) and tissue factor activity (OR 1.019, 95%CI 1.004-1.035) were risk factors, and high density lipoprotein (OR 0.008, 95%CI 0-0.478) and activated protein C ratio (OR 0.001, 95%CI 0-0.011) were protective factors for coronary heart disease. Conclusion There are activated extrinsic coagulation, lower response to activated protein C and damaged or activated endothelium function in SAP and ACS patients, plasma TFc may be a predictor for onset of ACS, plasma FVIIa and TFc are correlated with CHD. Part II Study on Protein Markers of Thrombosis in Coronary Heart DiseaseObjective To detect the plasma proteomic patterns in acute myocardial infarction (AMI) patients and controls, screen specific biomarkers and build diagnostic models by Surface-enhanced laser desorption/ionization time-of-flight mass spectrometer (SELDI-TOF-MS) and weak cation exchange protein chip (CM10). Methods 77 patients with acute myocardial infarction admitted to department of cardiology in Wuhan Union Hospital, 60 healthy controls and 32 patients with acute cerebral infarction (ACI) were enrolled from January to July 2006. Acute myocardial infarction group and control groups were matched for age, sex and sampling time. Protein chips combined with human plasma were placed in SELDI-TOF-MS for detecting protein profiling, analysis of the total experiment data was implemented by the Zhejiang University Cancer Institute-ProteinChip Data Analysis System (ZUCI-PDAS) for finding out discrepancy protein and building diagnostic models. Results A pattern composed five protein peaks (m/z: 3321.6, 16099.3, 8058.7, 15933.0 and 16286.7, respectively) with a specificity of 95% and a sensitivity of 88.3% was selected based on their collective contribution to the optimal separation between patients with AMI and healthy controls; A pattern composed seventeen protein peaks (m/z: 6682.6, 6485.6, 3321.6, 16682.9, 16297.7, 7977.5, 3424.7, 4821.1, 11748.7, 4775.3, 8977.3, 9094.8, 4361.4, 11479.9, 2941.1, 11399.7 and 2462.3, respectively) with a total accuracy of 100% was selected based on their collective contribution to the optimal separation between patients with AMI and patients with ACI. Conclusion Plasma proteomic profiling with SELDI-TOF-MS and ProteinChip technologies provides high sensitivity and specificity in discriminating patients with AMI and controls, and the discovered protein peaks might show great potential for early diagnosis of AMI. Part III Study on Gene Markers of Thrombosis in Coronary Heart Disease by Multi-Analyte Suspension Array technologyObjective Coronary heart disease is caused by multiple factors, which also include genetic factors. The main objective of this study was to detect 7 gene polymorphisms in acute myocardial infarction (AMI) patients by liquid gene chip technology (MASA technology), and positive findings were verified, so as to find out validity of MASA technology in detecting gene variations. Methods 34 patients with acute myocardial infarction admitted to department of cardiology in Wuhan Union Hospital from January to July 2006 were enrolled in this trail. DNA was amplified by PCR technology after being extracted, nucleic acid probes of 7 gene variations (FV Leiden G/A mutation, Fbg Beta -455 G/A mutation, Fbg Beta -148 C/T mutation, Fbg Beta 448 G/A mutation, FVII -323 10bp deletion/insertion mutation, prothrombin 20210 G/A mutation and MTHFR 677 C/T mutation) were combined with microspheres (beads) by covalent bonds, the latter were coded with specified fluorescence, PCR products was added after various beads were mixed, the mixture was detected by Luminex 100? Multi-Analyte Suspension Array, finally, positive findings were verified by Sanger dideoxynucleotide chain termination. Results There were 19 patients with gene variations, including 4 patients with FV Leiden mutation, 14 patients with MTHFR 677 C/T mutation, 5 patients with fibrinogenβgene 448 G/A mutation, among them there were 1 patient with FV Leiden and Fbgβ448G/A mutation, 1 with FV Leiden and MTHFR677 C/T mutation, 2 with MTHFR677 C/T mutation and Fbgβ448G/A mutation, the total accuracy of MASA in positive findings was 73.9%. Conclusion Liquid gene chips technology is feasible in detecting gene variations in AMI patients, nevertheless, the MASA technology needs to improve.更多还原