慢性血栓栓塞性肺动脉高压的病理生理机制研究及心肺运动试验的预测作用

发布时间：2018-05-01 14:41

本文选题：慢性血栓栓塞性肺动脉高压 + 铁代谢　；参考：《北京协和医学院》2015年博士论文

【摘要】：一、研究背景慢性血栓栓塞性肺动脉高压(Chronic thromboembolic pulmonary hypertension, CTEPH)在肺动脉高压分类中属于第四大类。急性肺血栓栓塞症(Acute pulmonary thromboembolism, APE)患者5年内发生CTEPH的累积发病率约为0.4%-5.1%。这一部分患者发生CTEPH的病理生理机制目前不明确。CTEPH的病理特征为肺动脉内不能消融的血栓继发纤维化、机化。既往有多项研究显示：1、铁代谢紊乱与血栓性疾病密切相关。2、病理研究显示CTEPH患者的非阻塞血管的远端动脉也发生了与特发性肺动脉高压(pulmonary arterial hypertension, PAH)相同的丛状病变。基因突变是特发性PAH及可遗传性PAH的重要发病机制,目前已发现多个基因突变与PAH发病有关,CTEPH患者是否存在PAH相关的基因突变目前尚不清楚。3、差异蛋白质组学研究是明确疾病发病机制、寻找疾病特异的蛋白质分子的良好方法。4、及早发现CTEPH的存在并给予积极治疗有望改善患者预后。因此本课题从铁代谢、基因突变和蛋白质组学三个方面探讨CTEPH可能的发病机制；并以心肺运动试验评估APE后不同疾病状态患者的表现,以寻找预测CTEPH的敏感因子。二、研究内容第一部分铁代谢在CTEPH发病过程中作用的研究1、研究目的通过比较CTEPH患者和急性PE事件后痊愈的患者铁代谢情况,明确铁代谢是否与CTEPH有关。2、研究方法2.1研究对象：2013年4月至2014年3月在阜外医院肺血管病科住院的明确诊断的CTEPH患者和慢性PE患者。2.2实验室检测：收集空腹血浆,检测血浆自由铁浓度、铁蛋白、可溶性转铁蛋白受体(sTfR)、hepcidin-25、肿瘤坏死因子-α (TNF-α)、白介素-6(IL-6)、丙二醛(MDA)水平。2.3统计学分析：连续数据两组间比较采用student's t检验或Mann-Whitney U检验；分类变量的两组间比较采用卡方检验。非结合铁离子浓度与hepcidin-25、 sTfR,铁蛋白、sTfR/铁蛋白比值、TNF-α,IL-6、MDA间的相关关系采用Spearman相关系数表示。hepcidin-25浓度与上述各检测指标的关系采用Spearman相关系数表示。Logistic回归模型分析CTEPH与上述各检测指标的关系。3、研究结果血浆非结合铁离子浓度在CTEPH组与慢性PE对照组无明显差别。两组间hepcidin-25、sTfR、TNF-α、MDA水平也无显著差别。CTEPH组的IL-6显著高于对照组。hepcidin-25水平与sTfR浓度呈负相关(Spearman's r=-0.438, p 0.001); hepcidin-25水平与铁蛋白呈正相关(Spearman'sr=0.503, p0.001).血浆非结合铁离子浓度与hepcidin-25、sTfR、铁蛋白、sTfR/铁蛋白比值、TNF-β、 IL-6、MDA均无显著相关(见表1-3).单变量logistic回归分析显示,非结合铁离子浓度、sTfR、铁蛋白、sTfR/铁蛋白比值、TNF-α、IL-6、MDA均与CTEPH发病无明显关系。4、第一部分研究结论铁代谢水平与CTEPH发病无明确相关。CTEPH患者和慢性PE患者的机体sTfR水平及铁蛋白水平均在正常范围。第二部分CTEPH患者的基因突变研究1、研究目的通过比较CTEPH患者和有PE病史而无PAH的患者的基因变异情况,目的是明确PAH相关的基因突变是否参与了CTEPH的发病。2、研究方法2.1研究对象：49名CTEPH患者和17名有PE病史而无PH的对照组患者。2.2基因突变检测：收集血液标本,提取全基因组DNA。直接测序并与参考序列比对检测PAH相关的基因：BMPR2、ACVRL1、ENG、SMAD9、CAV1、KCNK3、CBLN2。多重连接探针扩增(MLPA)方法检测片段重排突变。点突变对基因功能的影响以PolyPhen-2软件进行预测。2.3两组间的基因突变频率比较采用Fisher's检验。采用卡方检验明确基因型分布是否服从Hardy-Weinberg平衡定律。3、研究结果共66个样本进行了基因变异的分析,包括49名CTEPH患者及17名PE后无PAH的患者。CBLN2的CDS1因技术原因PCR扩增失败。共发现40个基因突变位点,包括31个点突变和9个片段重排。13个位点很可能造成功能破坏,2个突变位点可能造成破坏,2个突变位点为良性突变。3名研究对象有多处的点突变。CTEPH患者的PAH相关基因的非同义突变比率大于对照组(CTEPH组25/49(51%)vs.对照组3/17(18%),p=0.022)。共发现12个在dbSNP数据库中已有记录的单核苷酸多态性(single nucleotide polymorphism, SNP)。比较了两组间的基因型及等位基因频率后,发现SNP rs3739817和SNP rs55805125与CTEPH有关。4、第二部分研究结论中国汉族CTEPH患者中较高的PAH致病基因的突变频率可能与CTEPH发病有关。第三部分CTEPH患者的蛋白质组学研究1、研究目的从CTEPH患者的肺动脉内膜切除组织中进行差异蛋白质对比,以寻求发病过程中的“特异蛋白质分子”或“特异的信号转导通路”。2、研究方法2.1实验分组：1)CTEPH患者行肺动脉血栓内膜切除术中取得的增生内膜组织提取蛋白质的等份混合物；2)培养的人肺动脉内皮细胞、人肺动脉平滑肌细胞、人肺成纤维细胞,收集并鉴定后提取蛋白质的等份混合物。2.2两组蛋白质混合物以高效液相色谱(high-performance liquid chromatography, HPLC)分离后作色质联用进一步分离、鉴定。2.3两次均分离鉴定到的蛋白质在两组间比较,取得病变组织特异表达的蛋白质谱作进一步的G0和KEGG生物信息学分析。3、研究结果组织特异性的蛋白质为679个。由特异表达蛋白质的生物学过程富集结果显示,特异表达蛋白质主要参与了损伤反应、免疫反应、炎症反应、补体激活及血液凝固等生物过程。特异表达蛋白质主要构成细胞外基质、血小板、分泌颗粒、脂蛋白复合体以及囊泡等细胞组分。特异表达蛋白质主要发挥了与糖类结合、酶抑制剂激活以及与钙离子结合等功能。富集于补体及凝血通路、系统性红斑狼疮通路、ECM与受体结合通路、细胞粘附分子通路、Fc epsilon RI信号通路、白细胞跨内皮迁移通路、朊病毒疾病通路(CCL5,补体C1qa, C1QB, C1QC, C6, C7, C8A, C8B, C8G)、病毒性心肌炎、Fc gamma R介导的胞吞通路等信号通路。4、第三部分研究结论此部分CTEPH患者肺动脉血栓内膜的蛋白质组学研究得到679个病变组织特异的蛋白质。特异表达蛋白质主要参与了损伤反应、免疫反应、炎症反应、补体激活及血液凝固等生物过程。富集于补体及凝血通路、系统性红斑狼疮通路、ECM与受体结合通路、细胞粘附分子通路、Fc epsilon RI信号通路、白细胞跨内皮迁移通路等信号通路。第四部分心肺运动试验在预测CTEPH中的作用1、研究目的在这部分研究中,我们评估了APE存活者三种不同的预后状态,以期发现敏感的心肺运动试验的疾病监测指标。2、研究方法2.1研究对象：自2011年1月至2014年5月间连续入选符合标准的,在我科住院的66名CTEPH患者、44名慢性PE患者、15名PE痊愈患者。慢性PE, PE痊愈患者以及3例CTEPH来自2011年1月起始的中国肺栓塞注册登记研究(2011BA11B17).36名健康成年人作为对照组。2.2心肺运动试验：在医生监督下进行症状限制的功率递增式直立踏车运动试验。先进行3分钟静息数据采集后开始3分钟的无负荷踏车运动,继之3分钟的持续功率递增直至最大耐受量。气体交换采用逐次呼吸测量系统：功率递增幅度的选择根据对患者病史包括日常运动量和运动强度、体格检查和肺功能状况综合决定。递增方案：CTEPH组、CPE组为10～15 W/min,对照组为10～30 W/min。2.3统计分析：连续数据组间比较采用方差分析；分类变量的组间比较采用卡方检验。Pearson或Spearman相关分析临床数据与VE/VC02斜率、VD/VTphy、最低点VE/VCO2比值。多变量线性回归分析明确CPET中的独立预测CTEPH的指标。受试者工作曲线(receiver operator characteristic curve, ROC)分析预测效力。以ROC曲线的最佳截点分类待测数据,应用单变量和多变量logistic回归分析建立预测模型。3、研究结果CTEPH患者的最低点VE/VC02比值比慢性PE组和PE痊愈组明显增高(43.4L/min vs 29.9 L/min vs 27.1 L/min, p0.005); VE/VC02斜率(48.4 L/min/L/min vs 29.9 L/min/L/min vs 28.0 L/min/L/min, p0.005)及摄氧效率平台(OUEP) (37.1 L/min vs 27.0 L/min vs 25.2 L/min, p0.005)也显著高于慢性PE组和PE痊愈组。Logistic回归分析显示,最低点VE/VC02比值≥34.35 L/min是预测CTEPH的最强因子(OR 159.0,95% CI 36.0-702.3, p0.001)。慢性PE组的最低点VE/VC02比值较对照组高(29.9 L/min vs 26.5 L/min, p0.05),但最低点VE/VC02比值在痊愈组和对照组无差别。在多元线性回归分析中,基于肺通气／灌注显像评价的血管阻塞率(PVO)是慢性PE组和痊愈组的通气效率指标的独立预测因子。4、第四部分研究结论4.1CTEPH患者的通气效率降低；4.2最低点VE/VC02比值是预测CTEPH的最佳指标；4.3通气效率随急性PE恢复而改善。三、结论1、铁代谢水平与CTEPH发病无明确相关。CTEPH患者和慢性肺栓塞患者的机体sTfR水平及铁蛋白水平均在正常范围。2、中国汉族CTEPH患者中较高的PAH致病基因的突变频率可能与CTEPH发病有关。3、CTEPH患者肺动脉血栓内膜切除的组织特异表达蛋白质主要参与了损伤反应、免疫反应、炎症反应、补体激活及血液凝固等生物过程。富集于补体及凝血通路、系统性红斑狼疮通路、ECM与受体结合通路、细胞粘附分子通路、Fc epsilonRI信号通路、白细胞跨内皮迁移通路等信号通路。4、心肺运动试验是评估肺栓塞预后的良好方法,最低点VE/VC02比值是预测CTEPH的最佳指标。
[Abstract]:1. Background chronic thromboembolic pulmonary hypertension (Chronic thromboembolic pulmonary hypertension, CTEPH) is of fourth major categories in the classification of pulmonary hypertension. The cumulative incidence of CTEPH in patients with acute pulmonary thromboembolism (Acute pulmonary thromboembolism, APE) within 5 years is about 0.4%-5.1%. in this part of the patients. The pathophysiological mechanism of PH is not clearly defined as the pathological features of.CTEPH in the pulmonary artery that can not be melted with secondary fibrosis and machine. 1, a number of previous studies have shown that iron metabolic disorders are closely related to thrombotic diseases and.2. Pathological studies show that the distal arteries of the non blocking vessels of the CTEPH patients also occur with the idiopathic pulmonary hypertension Pulmonary arterial hypertension (PAH) is the same plexiform lesion. Gene mutation is an important pathogenesis of idiopathic PAH and hereditary PAH. Multiple gene mutations have been found to be associated with the pathogenesis of PAH. The existence of PAH related gene mutations in CTEPH patients is not yet clear of.3. Differential proteomics research is a clear disease pathogenesis. .4, a good method for searching for disease specific protein molecules, and early detection of the presence of CTEPH and active treatment are expected to improve the prognosis of the patients. Therefore, this subject studies the possible pathogenesis of CTEPH from three aspects of iron metabolism, gene mutation and proteomics, and the assessment of patients with different disease status after APE by cardiopulmonary exercise test. To find a sensitive factor for predicting CTEPH. Two, Part 1 of the study, Part 1, study of the role of iron metabolism in the pathogenesis of CTEPH. The purpose of this study was to determine whether iron metabolism was associated with CTEPH.2 by comparing the iron metabolism in patients with CTEPH and acute PE events, and method 2.1: April 2013 to March 2014. A clear diagnosis of CTEPH patients and chronic PE patients in the Fuwai Hospital of the Fuwai Hospital: collecting fasting plasma, detecting plasma free iron concentration, ferritin, soluble transferrin receptor (sTfR), hepcidin-25, tumor necrosis factor - alpha (TNF- alpha), interleukins -6 (IL-6), and malondialdehyde (MDA) level:.2.3 statistics analysis: The two groups of continuous data were compared with student's t test or Mann-Whitney U test; the two groups of classified variables were compared with chi square test. The correlation between the concentration of non binding iron ions and hepcidin-25, sTfR, ferritin, sTfR/ ferritin ratio, TNF- a, IL-6, MDA was carried out by Spearman correlation coefficient to indicate the.Hepcidin-25 concentration and the above tests. The relationship between measurement index and Spearman correlation coefficient was used to express.Logistic regression model to analyze the relationship between CTEPH and the above indexes.3. The results of the study showed no significant difference between the concentration of non binding iron ions in the CTEPH group and the chronic PE control group. There was no significant difference between the two groups of hepcidin-25, sTfR, TNF- alpha and MDA, and the IL-6 significantly higher than that of the.CTEPH group was higher than that of the pair. The level of.Hepcidin-25 was negatively correlated with the concentration of sTfR (Spearman's r=-0.438, P 0.001); hepcidin-25 level was positively correlated with ferritin (Spearman'sr=0.503, p0.001). There was no significant correlation between the concentration of non binding iron ions in plasma and hepcidin-25, sTfR, ferritin, sTfR/ ferritin ratio, TNF- beta, etc. (see table 1-3). The analysis showed that the concentration of non binding iron ions, sTfR, ferritin, sTfR/ ferritin ratio, TNF- a, IL-6, MDA were not significantly related to the pathogenesis of CTEPH.4. The first part of the study concluded that the level of iron metabolism was not clearly related to the pathogenesis of CTEPH and the sTfR level and the level of ferritin in the patients with.CTEPH and chronic PE were in the normal range. Second part CTEPH. Gene mutation study 1, the purpose of the study was to compare the genetic variation of CTEPH patients and patients with a history of PE without PAH. The purpose of the study was to determine whether PAH related mutations were involved in the pathogenesis of.2 in CTEPH. Study methods 2.1 subjects: 49 CTEPH patients and 17 patients with PE history without PH control group, the.2.2 gene mutation test. Test: collect the blood samples, extract the whole genome DNA. direct sequencing and compare with the reference sequence to detect PAH related genes: BMPR2, ACVRL1, ENG, SMAD9, CAV1, KCNK3, CBLN2. multiple connection probe amplification (MLPA) method for detecting fragment rearrangement mutation. The effect of point mutation on gene function is used to predict the genes between two groups of.2.3. The mutation frequency was compared with the Fisher's test. A chi square test was used to determine whether the genotype distribution was subject to the Hardy-Weinberg equilibrium law.3. The results of the study were analyzed in 66 samples, including 49 CTEPH patients and 17 PE without PAH for.CBLN2 CDS1 due to the failure of PCR amplification. A total of 40 gene mutations were found. Points, including 31 point mutations and 9 fragment rearrangements,.13 sites are likely to cause functional damage, 2 mutation sites may cause damage, 2 mutation sites are benign.3 names, and there are multiple point mutations in.CTEPH patients with PAH related genes more non synonymous mutations than the control group (CTEPH group 25/49 (51%) vs. control group 3/17 (18%), p= 0.022). A total of 12 single nucleotide polymorphisms (single nucleotide polymorphism, SNP), which were recorded in the dbSNP database, were found. After comparing the genotype and allele frequencies between the two groups, the SNP rs3739817 and SNP rs55805125 were found to be associated with CTEPH.4. The second part studies the higher pathogenic genes in the Chinese Han patients. The mutation frequency may be associated with the incidence of CTEPH. Third part of the proteomics study in partial CTEPH patients 1. The purpose of the study was to compare protein contrast in the pulmonary endarterectomy tissues of patients with CTEPH in order to seek "specific protein molecules" or "specific signal transduction pathways" in the pathogenesis of.2, and the study method 2.1 group. 1) an equal mixture of proteins extracted from the intima tissue of the CTEPH patients during the endarterectomy of pulmonary artery thrombosis; 2) the cultured human pulmonary artery endothelial cells, human pulmonary artery smooth muscle cells, human lung fibroblasts, and after collecting and identifying the equivalent mixture of protein.2.2 two groups of protein mixtures for high performance liquid chromatography After the high-performance liquid chromatography (HPLC) separation, the color composition was further separated, and the protein identified by the.2.3 two times was compared between the two groups. The protein mass specific expressed by the pathological tissue was obtained for further G0 and KEGG bioinformatics analysis.3, and the results of the study were 679 of the specific proteins. Biological process enrichment results show that specific protein mainly participates in biological processes such as injury reaction, immune response, inflammatory reaction, complement activation and blood coagulation. Specific proteins mainly consist of extracellular matrix, platelets, secretory particles, lipoprotein complexes and vesicles. The proteins expressed mainly in combination with carbohydrates, activation of enzyme inhibitors and binding with calcium ions. Enriched in complement and coagulation pathways, systemic lupus erythematosus pathway, ECM and receptor binding pathway, cell adhesion molecule pathway, Fc epsilon RI signaling pathway, white fine cell transendothelial pathway, prion disease pathway (CCL5, supplementation) C1qa, C1QB, C1QC, C6, C7, C8A, C8B, C8G), viral myocarditis, and Fc gamma R mediated endocytosis pathway and other signaling pathways. The third part studies the proteomic study of the pulmonary artery intima of the pulmonary artery of the patients. The specific protein is mainly involved in the damage response and immunization. Biological processes such as reaction, inflammatory response, complement activation and blood coagulation. Enrichment in complement and coagulation pathways, systemic lupus erythematosus pathway, ECM and receptor binding pathway, cell adhesion molecular pathway, Fc epsilon RI signaling pathway, and leucocyte transendothelial migration pathway. The fourth part cardiopulmonary exercise test in predicting CTEPH 1, in this part of the study, we evaluated three different prognostic states of APE survivors in order to find the disease monitoring index of the sensitive cardiopulmonary exercise test.2. Methods 2.1 research subjects: 66 CTEPH patients hospitalized in our department from January 2011 to May 2014 and 44 chronic PE patients hospitalized in our department. 15 PE patients, chronic PE, PE healers, and 3 cases of CTEPH from the January 2011 Chinese pulmonary embolism registration study (2011BA11B17).36 healthy adults as the control group.2.2 cardiopulmonary exercise test: a power incremental treadmill exercise test under the supervision of a doctor under the supervision of a doctor. An advanced line of 3 minutes resting The 3 minute load free treadmill started after the data collection, followed by a 3 minute continuous power increase to the maximum tolerance. Gas exchange using a successive respiratory measurement system: the choice of power increasing amplitude was based on the patient's medical history including daily movement and exercise intensity, physical examination and lung function status. An incremental scheme: CT The EPH group, CPE group was 10~15 W/min, and the control group was 10~30 W/min.2.3 statistical analysis: the continuous data group was compared with the analysis of variance; the group comparison of the classified variables used the chi square test.Pearson or Spearman correlation analysis clinical data with the VE/VC02 slope, VD/VTphy, and the lowest VE/ VCO2 ratio. Multivariate linear regression analysis made clear CPET in the CPET. Receiver operator characteristic curve (ROC) was used to predict the effectiveness of CTEPH independently. The optimal section of the ROC curve was used to classify the data, and the single variable and multivariable logistic regression analysis was used to establish the prediction model.3. The results of the study results of the lowest VE/VC02 ratio of the CTEPH patients were better than those of the chronic PE group and the recovery. The group was significantly higher (43.4L/min vs 29.9 L/min vs 27.1 L/min, P0.005), VE/VC02 slope (48.4 L/min/L/min vs 29.9 L/min/L/min vs 28 L/min/L/min, 37.1) and oxygen uptake efficiency platform (37.1) was also significantly higher than that of the chronic group and the recovery group. The VC02 ratio of more than 34.35 L/min was the strongest factor for predicting CTEPH (OR 159.0,95% CI 36.0-702.3, p0.001). The lowest VE/VC02 ratio of the chronic PE group was higher than that of the control group (29.9 L/min vs 26.5), but there was no difference between the recovery group and the control group. The rate of vascular obstruction rate (PVO) was an independent predictor of the ventilation efficiency index in the chronic PE group and the healing group. The fourth part of the study concluded that the ventilation efficiency of the 4.1CTEPH patients was reduced; the 4.2 lowest VE/VC02 ratio was the best indicator of the prediction of CTEPH; the 4.3 ventilation efficiency was improved with the acute PE recovery. Three, conclusion 1, the iron metabolism level and CTEPH pathogenesis. The level of sTfR and ferritin in the patients with.CTEPH and chronic pulmonary embolism were in a normal range of.2. The mutation frequency of the higher PAH pathogenic gene in Chinese Han CTEPH patients may be associated with the pathogenesis of CTEPH, and the tissue specific protein expressed in the patients with pulmonary artery thrombosis in CTEPH patients is mainly involved in the injury reaction. Biological processes such as immune response, inflammatory response, complement activation and blood coagulation, enrichment in complement and coagulation pathways, systemic lupus erythematosus pathways, ECM and receptor binding pathways, cell adhesion molecules pathway, Fc epsilonRI signaling pathway, leucocyte transendothelial migration pathway,.4, cardiopulmonary exercise test is a good prognosis assessment of pulmonary embolism Good method, the lowest VE/VC02 ratio is the best indicator for predicting CTEPH.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R544.1

【共引文献】