【作者】 张小平；

【导师】 胡豫；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 血栓性疾病(Thrombotic diseases)是涉及临床各科的一大类疾病。动脉粥样硬化及其血栓栓塞性并发症在我国和西方国家均已成为人口死亡与致残的第一位原因，且呈逐年上升趋势。血栓的发生是一个较为长期的过程，患者就医时，血栓往往已经形成并导致血管栓塞。心脏和脑等对缺血十分敏感的器官，发生栓塞后治疗效果不佳，故血栓性疾病严重危害人类健康。如能在血栓发生的早期即进行诊断并及时采取干预措施，则能够大大降低血栓栓塞性疾病的危害。目前，血栓性疾病的确诊主要依靠血栓形成部位的影像学证据。临床上主要以D-二聚体(d-dimer)、活化部分凝血酶原时间(activated partial thromboplastin time，APTT)、凝血酶原时间(prothrombin time，PT)、纤维蛋白原(fibrinogen，Fbg)含量等作为血栓性疾病的初步筛查项目，其敏感度和准确性尚无统一的临床结论。血栓性疾病发病机制极其复杂，在血栓形成早期，或血栓尚未形成时，上述筛查指标可能出现阴性结果，往往延误正确判断；而当阳性结果出现时，疾病往往已进展到中晚期，失去了最有效的治疗时机。因此，在血栓病高危人群中，对血栓发生危险性进行全面的评估及早期诊断血栓形成已成为当今血栓病研究领域的热点。蛋白质-飞行时间质谱技术(Time of flight mass spectrometry，TOF-MS)是蛋白质组学中一种大规模、高通量的分子研究方法，与生物信息学和基因功能实验等相结合已经在揭示疾病发生的分子机制领域中发挥越来越重要的作用。本试验采用表面增强激光解析/电离飞行时间质谱仪(surface enhanced laser desorption/ionization time offlight mass spectrometry，SELDI-TOF-MS)及蛋白质生物芯片技术探索性研究动脉、静脉血栓形成患者的血浆蛋白质指纹图谱，旨在发现能够区分血栓病与对照组的高敏感、高特异的蛋白质生物标志物，为开发新的可用于诊断血栓形成的特异性标记物提供实验依据。同时，结合一系列与凝血、抗凝及纤溶系统相关的液态基因芯片检测平台及相关生化指标检测，通过临床研究，为发现血栓形成建立快速、准确的早期诊断综合体系。第一部分急性脑梗死凝血功能变化及其危险因素评估的回顾性研究目的：已有研究表明，血液中凝血、抗凝或纤溶蛋白水平的改变与脑血管疾病的发生明显相关。但较少研究综合考虑其在脑血管疾病中的改变及作用。在本次研究中，我们系统探讨了其中的16个凝血指标、血脂水平、以及其他可能的危险因素与急性脑梗死的关系。方法：武汉协和医院急性脑梗死(Acute Cerebral Infarction，ACI)住院患者50例，住院对照54例。所有研究对象均在入院第二天清晨空腹采取肘静脉血。血浆蛋白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 factorⅦ，FⅦa)、凝血因子Ⅶ抗原(factorⅦantigen，FⅦAg)、P-选择素(p-selectin)、组织型纤溶酶原激活剂(tissue-type plasminogen activator，t-PA)及纤溶酶原激活剂抑制物-1(plasminogen activator inhibitor-1，PAI-1)水平均由ELISA试剂盒检测；组织因子活性(tissue factor activity，aTF)由发色底物法检测；全自动凝血分析仪(SysmexCA-7000，日本)检测活化蛋白C(activated protein C，APC)比率、活化部分凝血活酶时间(activated partial thromboplastin time，APTT)、凝血酶原时间(prothrombin time，PT)、凝血酶时间(thrombin time，TT)、纤维蛋白原(fibrinogen，Fbg)以及D-二聚体(d-dimer)；检验科自动生化分析仪(日立7170A，东京，日本)检测血脂水平。结果：疾病组血浆凝血酶调节蛋白、纤维蛋白原水平及组织因子活性显著高于对照组(P＜0．001，P＜0．01，P＜0．05)；而高密度脂蛋白水平显著低于对照组(P＜0．01)。多变量逻辑回归分析表明：高血压的存在，血浆高水平的凝血酶调节蛋白、纤维蛋白原及组织因子活性与急性脑梗死的发病明显相关(比值比[odds ratio，OR]=143．74，P＜0．001；OR=2．05，P＜0．05；OR=2．09，P＜0．05；OR=1．02，P＜0．05)。结论：高血压，血浆凝血酶调节蛋白、纤维蛋白原水平及组织因子活性的升高是急性脑梗死的独立危险因素。第二部分急性深静脉血栓形成危险因素的评估目的：观察急性下肢深静脉血栓形成(deep venous thrombosis，DVT)患者凝血功能变化及评估其危险因素。方法：全自动凝血分析仪(Sysmex CA-7000，日本)检测62例急性下肢DVT患者和70例健康对照者血浆活化部分凝血活酶时间(activated partial thromboplastin time，APTT)、凝血酶原时间(prothrombin time，PT)、凝血酶时间(thrombin time，TT)、纤维蛋白原(fibrinogen，Fbg)及D-二聚体(d-dimer)水平；检验科自动生化分析仪(日立7170A，东京，日本)检测高密度脂蛋白胆固醇(high-density lipoprotein cholesterol，HDL-C)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol，LDL-C)、总胆固醇(total cholesterol，TC)及甘油三酯(triglyceride，TG)水平；并通过二分类逻辑回归分析回顾性研究所有患者的临床资料。结果：(1)DVT组血浆APTT、PT、TT、D-二聚体、Fbg水平及D-二聚体与Fbg比值(D/F值)都明显高于健康对照组，差异均有统计学意义(所有P＜0．01)；(2)DVT组和对照组血浆D-二聚体与Fbg水平之间均存在正相关性(r=0．475，P＜0．01；r=0．564，P＜0．01)；(3)逻辑回归分析表明：急性下肢DVT的发生与患者存在高血压，血浆纤维蛋白原水平的升高明显相关(比值比[odds ratio，OR]=24．99，P＜0．01；OR=4．346，P＜0．01)。结论：高血压和升高的血浆纤维蛋白原是急性下肢DVT的独立危险因素。第三部分急性血栓性疾病血浆蛋白质指纹图谱研究实验一急性脑梗死血浆潜在标志物的蛋白质组学研究目的：既往研究表明，并不存在单一的生化指标可用于急性脑梗死(Acute CerebralInfarction，ACI)的常规诊断。本实验运用表面增强激光解吸/电离-飞行时间质谱(Surface-enhanced laser desorption/ionization time of flight mass spectrometry，SELDI-TOF-MS)技术筛选急性脑梗死患者血浆中潜在的特异标志物。方法：入选急性脑梗死患者32例，健康对照者60例。采用SELDI-TOF-MS及弱阳离子交换芯片阵列技术(weak cation exchange CM10 ProteinChip arrays，CiphergenBiosystems)检测两组研究对象的血浆蛋白质质谱；并借助生物信息学工具(非线性的支持向量机，nonlinear support vector machine)提出急性脑梗死的诊断模型，并运用留一交叉验证法(Leave-one-out cross validation)来评估该模型的判别效能。结果：由13个标志物组成的血浆蛋白质质谱诊断模型，能够最佳地区分脑梗死与正常对照组。该模型具有84．4％的敏感性和95．0％的特异性。结论：SELDI-TOF-MS及蛋白质芯片技术在急性脑梗死的诊断中具有潜在的应用前景。急性脑梗死的诊断可能依赖于一组生物标志物的结合。实验二急性心肌梗死及深静脉血栓形成血浆潜在标志物的蛋白质组学研究目的：本研究运用表面增强激光解吸/电离-飞行时间质谱(Surface enhanced laserdesorption/ionization time of flight mass spectrometry，SELDI-TOF-MS)技术来寻找血栓栓塞性疾病(动脉和静脉)的特异生物标志物。方法：我们筛查了69例血浆样本中的潜在生物标志物，其中包括20例特发性深静脉血栓形成(Deep vein thrombosis，DVT)、20例急性心肌梗死(Acute myocardialinfarction，AMI)患者以及29例既往无血栓栓塞疾病史的健康对照者。在蛋白质生物系统Ⅱc及SELDI-TOF-MS(Ciphergen Biosystems，Fremont，CA)上分析预先处理过的血浆样品。并运用铜离子活化的固定金属鳌合亲和层析芯片阵列(immobilized metalaffinity chromatography，IMAC-3)产生蛋白质组学的波谱，并用质荷比(mass to chargeratio，m/z)来表示。结果：质荷比分别为2667、5914及6890 Da的3个生物标志物组成一种诊断模型。该模型能够最好地区分AMI患者与正常对照者，具有100％的正确率。另外一种仅由一个生物标志物(m/z，5914 Da)组成的诊断模型，也能够完全将DVT患者与正常对照者区别开。进一步对AMI与DVT患者之间的分析表明，由质荷比分别为3418、5271、33378及68125 Da组成的判别模型具有82．5％的准确率。结论：在区分血栓病患者与健康对照者时，SELDI-TOF-MS及蛋白质芯片技术具有很高的敏感性及特异性。对血栓栓塞性疾病的早期诊断而言，血浆中所发现的生物标志物展现出较大的应用潜能。更多还原

【Abstract】 As a main category of illness,thrombotic diseases have close relationship with eachclinical department.Atherosclerosis and its thromboembolic complication has become thefirst top of causes for death and disability both in western countries and China.Theincidence is increasing year by year.Process of thrombogenesis is a long period of duration.When a patient visits his doctor,a thrombus has already formed leading to vascularembolism.Vital organs,including the heart and brain which are very sensitive to ischemia,have bad response to therapy after an event of embolism.Therefore,thrombotic diseasesseverely do harm to human well-being.If a diagnosis of thrombosis during the early stageof thrombogenesis is performed and interventional measures are taken in time,the harmfuleffect caused by thromboembolic diseases can be weakened to a great extent.At present,accurate diagnosis for thrombotic diseases mainly depends on imageological evidence at the regional location of thrombosis.In clinical practice,detection of plasma d-dimer,activated partial thromboplastin time （APTT）,prothrombintime （PT）,and level of fibrinogen is considered to be an initial screening proceedingwithout a positive conclusion on the sensitivity and specificity.The pathogenesis ofthrombotic diseases is very complicated.At the early stage of thrombosis or prior to theformation of a thrombus,negative results might be shown about the above-mentionedscreening markers,ultimately leading to delay in precise diagnosis.When positive findingsare indicated,the illness usually develops to be at an intermediate or advanced stage duringwhen the most effective treatment couldn’t be performed.Therefore,among high-riskgroup with thrombotic diseases,the popular research today in the field of thromboticdiseases is concentrated on allround assessment of risk and performance of diagnosis ofthrombosis at an early stage.For studies on proteomics,time-of-flight mass spectrometry （TOF-MS） is an extensiveand high-throughout research technique at molecular level.Application of TOF-MScoupled with bioinformatics and experiments on gene function are playing more and moreimportant roles in the field of bringing molecular mechanisms of a disease to light.Usingsurface enhanced laser desorption / ionization time of flight mass spectrometry（SELDI-TOF-MS） and protein biochips,exploratory studies were performed to drawplasma protein fingerprints for arterial and venous thrombosis in order to discover specificprotein biomarkers which could discriminate between patients with thrombotic diseasesand control subjects with high sensitivity and specificity and to provide experimentalevidence for developing new specific biomarkers used for diagnosis of thrombosis in thefuture.Meanwhile,coupled with detection of correlated biomarkers and application of aseries of platforms for liquid-phase gene chips related with coagulation,anticoagulation,and fibrinolytic systems,a fast,precise,and comprehensive system for early diagnosisshould be set up to find the presence of a thrombus based on clinical investigation. PartⅠRetrospective Study on Changes of Coagulation Function andEvaluation on Risk Factors for Acute Cerebral InfarctionObjective Several studies have indicated an association between changes ofcoagulation,anticoagulation,and fibrinolytic proteins and development of cerebrovasculardisease,but few reports referred to their roles together.In this report,we systematicallyinvestigated the relationship between sixteen coagulation markers of them and acutecerebral infarction,as well as considering the contribution of blood lipids and otherpossible risk factors.Methods Inpatients diagnosed for acute cerebral infarction （ACI） （n=50） andhospital controls （n=54） were recruited from Union Hospital.All blood samples werecollected from ulnar vein on the mornings of 2^nd day of hospitalization.Plasmaconcentrations of protein C （PC）,free proteinS （FPS）,total protein S （TPS）,thrombomodulin （TM）,activated FⅦ（FⅦa）,FⅦantigen （FⅦAg）,p-selectin （p-sel）,tissue-type plasminogen activator （t-PA）,and plasminogen activator inhibitor-1 （PAI-1）were assayed by ELISA kits;Activity of tissue factor （aTF） was analyzed by chromogenicactivity assay kits;Activated protein C （APC） ratio,activated partial thromboplastin time（APTT）,prothrombin time （PT）,thrombin time （TI）,fibrinogen （Fbg）,and D-dimer weredetected in an automated coagulation analyzer （Sysmex CA-7000,Japan）.Blood lipidswere detected using an automatic biochemical analyzer （Hitachi 7170A,Tokyo,Japan） at Department of Laboratory Medicine.Results Plasma levels of thrombomodulin,fibrinogen,and activity of tissue factorwere significantly higher in cases than in control subjects （P＜0.001,P＜0.01,and P＜0.05,respectively）;Concentration of high-density lipoprotein cholesterol （HDL-C） wassignificantly lower in cases than in controls （P＜0.01）.Multivariate logistic regressionanalysis showed that hypertension and high plasma levels of thrombomodulin,fibrinogen,and aTF were significantly associated with presence of ACI （odds ratio [OR],143.74,P＜0.001;OR,2.05,P＜0.05;OR,2.09,P＜0.05;OR,1.02,P＜0.05,respectively）.Conclusion Our findings indicate that hypertension and elevation of plasmathrombomodulin,fibrinogen,and aTF are independent risk factors for ACI. PartⅡAssessment of Risk Factors for Acute Deep Venous ThrombosisObjective To observe the changes of coagulation function in patients with acutelower-limb deep venous thrombosis （DVT） and evaluate the risk factors for DVT.Methods Plasma APTT,PT,TT,fibrinogen （Fbg）,and D-dimer were detected by anautomated coagulation analyzer in 62 acute lower-limb DVT patients and 70 controlsubjects;And retrospective studies on the clinical data of all patients were done by binarylogistic regression analysis.Results （1） In DVT group,plasma APTT,PT,TT,D-dimer,and fibrinogen andD-dimer / fibrinogen ratio （D/F ratio） were higher when compared with control group;allof the differences were significant （all P＜0.01）;（2） There were positive correlationsbetween D-dimer and fibrinogen both in DVT and control groups （r= 0.475,P＜0.01;r=0.564,P＜0.01,respectively）;（3） Logistic analysis indicated that acute lower-limb DVTwas associated with the presence of hypertension and increased plasma level of fibrinogen（odds ratio [OR],24.99,P＜0.01;OR,4.346,P＜0.01,respectively）.Conclusions Hypertension and elevated plasma level of fibrinogen are independentrisk factors for acute lower-limb DVT. PartⅢStudy on Plasma Protein Fingerprints for Acute Thrombotic DiseasesExperiment 1 Proteomic study on plasma potentialbiomarkers for acute cerebral infarctionObjective Previous researches indicated that no single biologic marker was used inthe routine diagnosis of acute cerebral infarction （ACI）.In this experiment,surfaceenhanced laser desorption/ionization time of flight mass spectrometry （SELDI-TOF-MS）technology was used to screen potential specific biomarkers in plasma samples frompatients with ACI.Methods Plasma samples were drawn from 32 cases with ACI and from 60 healthycontrol subjects.Plasma proteomic profiling was detected by the SELDI-TOF-MStechnology coupled with weak cation exchange arrays （CM10 ProteinChip,CiphergenBiosystems）.A diagnostic pattern was introduced with the help of a bioinformatics tool（nonlinear support vector machine） and the method of leave-one-out cross validation wasused to estimate the discriminating power of this pattern.Results A differential pattern consisting of 13 biomarkers was selected based ontheir collective contribution to the optimal separation between patients with ACI andcontrol subjects with a sensitivity of 84.4% and specificity of 95.0%,respectively.Conclusion Plasma proteomic profiling with SELDI-TOF-MS and ProteinChiptechnologies shows potential in discriminating patients with acute cerebral infarction andcontrol subjects.Diagnosis of acute cerebral infarction should probably depend on the useof a panel of biomarkers. Experiment 2 Proteomic study on plasma potential biomarkers foracute myocardial infarction and deep vein thrombosisObjective The present study is concerned about searching for specific biomarkersfor thromboembolic （arterial and venous） diseases by the use of Surface-Enhanced LaserDesorption / Ionization Time-of-Flight Mass Spectrometry （SELDI-TOF-MS）.Methods We screened for potential biomarkers in 69 plasma samples,includingsamples from 20 patients with idiopathetic deep vein thrombosis （DVT）,20 patients withacute myocardial infarction （AMI）,and 29 healthy controls without a history ofthromboembolism.Pretreated plasma samples were analyzed on the Protein BiologySystem IIc plus SELDI-TOF-MS （Ciphergen Biosystems,Fremont,CA）.Proteomic spectraof mass to charge ratio （m/z） were generated by the application of plasma to immobilizedmetal affinity capture （IMAC-3） ProteinChip arrays activated with copper.Results A pattern of three biomarkers （m/z:2 667,5 914,and 6 890 Da,respectively）with a total accuracy of 100% was selected based on their collective contribution to theoptimal separation between patients with AMI and healthy controls.Another patternconsisting of only one biomarker （m/z:5 914 Da） could totally discriminate patients withDVT and control subjects.For further analysis between patients with AMI and those withDVT,a pattern of four biomarkers （m/z:3 418,5 271,33 378,and 68 125 Da,respectively）was selected with a total accuracy of 82.5%.Conclusions Plasma proteomic profiling with SELDI-TOF-MS and ProteinChiptechnologies provides high sensitivity and specificity in discriminating patients withthrombosis and healthy subjects.The discovered biomarkers might show great potential forearly diagnosis of thromboembolic diseases.更多还原