【摘要】：基于UHPLC-QTOF/MS的冠心病非靶向代謝組學分析及代謝標志物的探索目的:不同人群受到不同遺傳因素和環(huán)境危險因素相互作用后將最終影響共同的生物代謝過程,因此通過代謝組學手段從小分子代謝物水平的變化研究冠心病動脈粥樣硬化的病理形成機制已成為新的研究方向。本研究旨在通過非靶向代謝組學方法探索中國冠心病患者群體的血漿小分子代謝特點及其與疾病發(fā)生發(fā)展的關系。方法:經過嚴格的冠脈造影資料審查,從中國醫(yī)學科學院國家心血管病中心阜外心血管病醫(yī)院的795位通過篩選的患者中抽取300位患者建立了一個獨立的代謝組學研究隊列:150人嚴重冠脈狹窄的冠心病患者組(severe coronary heart disease,sCHD)和年齡與性別1:1匹配的150人冠脈造影正常對照組(angiographically normal controls,ANC)。我們將冠狀動脈造影顯示至少一支主支冠狀動脈管腔直徑縮窄≥80%定義為sCHD組,將經冠脈造影確認冠狀動脈管腔直徑縮窄≤ 20%定義為ANC組。抽取患者的靜脈血分離血漿,運用超高效液相色譜-飛行時間質譜聯(lián)用(UHPLC-QTOF/MS)的代謝組學檢測方法,從陽離子相和陰離子相兩種電離模式進行了血漿代謝組學分析。將篩選出的具有明確二級質譜定性信息的小分子代謝物分別加入多元線性回歸模型(P0.05),分析校正傳統(tǒng)冠心病危險因素(年齡、性別、體重指數(shù)、糖尿病、高血壓、高脂血癥、冠心病家族史、吸煙史)和檢測批次等因素。為了防止n次檢驗可能帶來的Ⅰ類錯誤,又進行了 Bonferroni correction校正(P8.2×10-5(0.05/611)),并使用logistic回歸模型分析了這些小分子代謝物與冠心病患病風險關聯(lián)的方向和程度。結果:除年齡和性別進行了匹配,冠心病傳統(tǒng)危險因素在嚴重冠脈狹窄的冠心病患者組皆顯著高于冠脈造影正常對照組。300個血漿樣本上機檢測后我們一共獲得611個(466個陽離子模式、145個陰離子模式)具有明確定性半定量信息的有效峰,多元線性回歸分析(P0.05)校正傳統(tǒng)冠心病危險因素和檢測批次后我們一共篩選出105個差異代謝物(其中21個實為未知代謝物)在兩組間血漿水平具有顯著性差異:主要包含51種磷脂、10種肉毒堿、8種游離脂肪酸類、5種膽汁酸代謝中間產物、3種氨基酸和7種未統(tǒng)一分類代謝物(吲哚、己酮酸、4-吡哆酸、水楊尿酸、咖啡因、睪酮、對甲氧基苯乙酸)。經 Bonferroni correction 校正(P8.2×10-5(0.05/611))從 105種差異代謝物中篩選出了 6種組間差異最為顯著的血漿代謝物(吡哆酸、棕櫚酸、亞油酸、脂酰肉毒堿(14:1)、石膽酸、磷脂酰甘油(20:3/2:0)),且這些代謝物皆表現(xiàn)出與冠心病患病風險顯著的正相關(oddsratio,OR值范圍2.79-7.24)。結論:本研究借助非靶向代謝組學方法獲得了中國冠心病患者群體的代謝指紋圖譜,并首次報道了吡哆酸、石膽酸和磷脂酰甘油(20:3/2:0)這三種與冠心病病理生理過程有強關聯(lián)的潛在生物標志物,為從整體水平探索冠心病動脈粥樣硬化發(fā)病機制及尋求新的治療途徑提供了依據(jù)。吡哆酸對血管內皮細胞增殖及單核巨噬細胞炎癥反應的影響目的:我們前期的研究通過血漿代謝組學檢測發(fā)掘了一種在嚴重冠脈狹窄的冠心病患者組和冠脈造影正常對照組之間存在顯著性差異的小分子代謝物吡哆酸，并且發(fā)現(xiàn)血漿吡哆酸每增加1%冠心病患病風險就增加4.879倍。為了進一步探討吡哆酸在動脈粥樣硬化形成進展過程中的作用，我們在細胞水平初步探索了吡哆酸可能對兩種冠狀動脈粥樣硬化病理形成緊密相關的細胞(人臍靜脈內皮細胞(HUVEC)，人單核細胞系(THP-1))產生的影響。方法:我們先后使用不同刺激濃度(20 nmol/L、50 nmol/L、100 nmol/L、200 nmol/L、500 nmol/L、1000 nmol/L、2000 nmol/L)的吡哆酸溶液刺激體外培養(yǎng)的人臍靜脈內皮細胞(HUVEC)、人單核細胞系(THP-1)，于不同刺激時點(12h24h48h和72h)通過細胞活力檢測分析吡哆酸對人臍靜脈內皮細胞產生的增殖影響，于吡哆酸刺激 48h 時通過實時熒光定量 PCR(Real-time quantitative Polymerase Chain ReactionqRT-PCR)的方法檢測白介素1β(IL-1β)、腫瘤壞死因子(TNF-α)和細胞粘附因子(ICAM)的表達變化以評估炎癥反應變化。結果:研究發(fā)現(xiàn)給予吡哆酸刺激48小時，人臍靜脈內皮細胞增殖活力開始發(fā)生了顯著下降且隨著吡哆酸濃度增加而逐漸降低2000nmol/L濃度刺激時最低可下降10.7%。給予吡哆酸刺激72小時，人臍靜脈內皮細胞增殖活力從刺激濃度20nmol/L起即表現(xiàn)出增殖活力顯著下降，隨著吡哆酸濃度增加而逐漸降低，1000nmol/L濃度刺激時最低可下降10.56%。低濃度的吡哆酸刺激沒有誘發(fā)單核細胞的炎癥反應，而當吡哆酸濃度為1000nmol/L和2000nmol/L時，THP-1細胞IL-1β、TNF-α和ICAM表達顯著高于空白對照組細胞，，IL-1β表達量最高升高到1.22倍、TNF-α的表達量最高升高到1.4倍、ICAM的表達量最高升高到1.74倍。結論:吡哆酸能夠誘導人臍靜脈內皮細胞出現(xiàn)增殖抑制、人單核細胞系炎癥因子(IL-1β、TNF-α、ICAM)表達上調，因此推測血漿吡哆酸水平升高與動脈粥樣硬化的形成、發(fā)展相關�；旌现萌胨幬锵疵撝Ъ芎徒饘俾阒Ъ苤委煻嗵幉∽児谛牟』颊叩倪h期療效和安全性評價目的:藥物洗脫支架(DES)有效降低了金屬支架置入后的再狹窄發(fā)生率及靶血管或靶病變的血運重建率,但其涂層藥物能夠導致靶血管內皮修復延遲以及致死性支架內血栓形成風險升高,引起了學界對DES使用安全性的關注與探討。因此針對冠狀動脈多處病變患者,術者們開始嘗試藥物洗脫支架和金屬裸支架混合置入(Hybrid支架)的介入治療策略,以期通過適度減少藥物支架置入數(shù)量來提高PCI治療安全性并獲得最佳的臨床效果。本研究旨在評估混合置入藥物洗脫支架(Hybrid支架)和金屬裸支架治療多處病變冠心病患者的遠期療效和安全性。方法:本研究入選了 2004年4月至2006年10月期間,在我院行擇期經皮冠狀動脈介入治療(PCI)的冠狀動脈多處病變患者共計6495例,并根據(jù)支架置入種類分為藥物洗脫支架和金屬裸支架混合置入組(Hybrid組,848例)和單純藥物洗脫支架置入組(5647例)。按1:1的比例進行傾向性評分匹配后,共823對患者最終納入研究。臨床隨訪資料包括術后30天、1年和2年的死亡、心肌梗死、靶病變血運重建、靶血管血運重建、主要心臟不良事件和支架內血栓形成。研究對比分析了兩組術后2年內的各種臨床事件的累積發(fā)生率差異,并通過Cox比例風險模型對臨床事件風險進行了評估。結果:經過傾向性評分匹配后COX比例風險模型分析顯示術后2年Hybrid支架治療組靶病變血運重建(風險比2.38,95%可信區(qū)間1.50-3.70)、靶血管血運重建(風險比1.61,95%可信區(qū)間1.15-2.27)以及主要心臟不良事件(風險比1.37,95%可信區(qū)間1.02-1.85)的風險顯著高于單純DES支架治療組,兩組間全因死亡、心肌梗死和全因死亡/心肌梗死差異無統(tǒng)計學意義。術后2年Hybrid支架治療組累積晚期血栓發(fā)生率顯著低于單純DES支架治療組(P=0.0112),而2年累積支架內其他血栓形成事件:肯定的血栓、肯定+可能血栓、早期血栓及極晚期血栓發(fā)生率無明顯差異。結論:對于冠狀動脈多處病變需置入多支架的患者而言,單純藥物洗脫支架置入的遠期有效性明顯優(yōu)于混合置入藥物洗脫支架和金屬裸支架,兩種支架置入策略安全性相當。冠狀動脈介入治療后圍術期心肌梗死風險模型及評分系統(tǒng)研究目的:既往國內外關于經皮冠狀動脈介入治療(PCI)后圍術期心肌梗死的發(fā)生風險并無系統(tǒng)、全面的研究,本研究旨在針對美國心血管介入學會(SCAI)制定的PMI定義,系統(tǒng)的篩選出圍術期心肌梗死的獨立危險因素,并建立PMI風險模型及評分系統(tǒng),用于全面評估多個危險因素共同作用下圍術期心肌梗死的風險。方法:本研究入選了 2013年1月至2013月12月間在中國醫(yī)學科學院阜外醫(yī)院成功行擇期PCI治療并具備圍術期心臟損傷標志物監(jiān)測資料的患者共3371例(3516次PCI治療)。根據(jù)患者術后是否發(fā)生SCAI所定義的圍術期心肌梗死(PMI),分為圍術期心肌梗死組和無圍術期心肌梗死組。篩選可能影響圍術期心肌梗死的危險因素:患者臨床基線資料、心肌損傷標志物等生化指標、冠狀動脈造影特點、介入操作特點等,納入Logistic多元回歸分析識別出圍術期心肌梗死的獨立危險因子并建立圍術期心肌梗死風險模型。根據(jù)各個危險因素對圍術期心肌梗死的危險度分配相應的分數(shù),形成PMI風險評分系統(tǒng)。通過Bootstrap方法從入選患者中內部驗證評分系統(tǒng)(樣本量n=3516,再抽樣次數(shù)B=1000),進一步驗證風險模型和評分系統(tǒng)的預測能力和有效性。結果:在所有成功實施經皮冠狀動脈介入治療的3371例患者患者中,有107例患者(3.1%)對應108次PCI治療發(fā)生了圍術期心肌梗死。通過多因素Logistic回歸分析發(fā)現(xiàn),影響圍術期心肌梗死的獨立危險因素有:年齡(比值比(OR)為1.037,95%置信區(qū)間(CI):1.016-1.058;P0.01)、多支血管治療(OR 為 1.697,95%CI:1.095-2.629;P=0.02)、至少一處分叉病變治療(OR 為 1.869,95%CI:1.213-2.878;P0.01)、靶病變總長度(OR為1.016,95%CI:1.009-1.024;P0.01)。由此建立的PMI評分系統(tǒng) ROC 曲線下面積為 0.71(95%CI:0.66-0.76),H-L p=0.5;經 Bootstrap 方法內部驗證得到PMI風險評分系統(tǒng)平均ROC曲線下面積為0.707,中位ROC曲線下面積為0.708;根據(jù)PMI評分的四分位數(shù),將前三個四分位Q1-Q3合并定義為非PMI高危組(對應風險評分10分)而第四四分位Q4定義為為PMI高危組(對應風險評分≥10分),圍術期心肌梗死風險在PMI高危組顯著高于非PMI高危組(P0.01)。結論:本研究全面系統(tǒng)的篩選出了影響圍術期心肌梗死的獨立危險因素:年齡、靶病變總長度、多支血管治療、至少一處分叉病變治療,并建立了簡便易用的PCI后圍術期心肌梗死風險評分,準確劃分出圍術期心肌梗死的高風險患者,為臨床決策提供客觀依據(jù)。
[Abstract]:Untargeted metabolomic analysis and exploration of metabolic markers for coronary heart disease based on UHPLC-QTOF/MS Objective: Different populations will eventually affect the common metabolic processes after interaction between different genetic and environmental risk factors, so the changes of small molecular metabolites in coronary heart disease atherosclerosis will be studied by metabonomics. The aim of this study was to explore the characteristics of plasma small molecule metabolism and its relationship with the occurrence and development of coronary heart disease in Chinese population by non-targeted metabonomics. An independent metabonomic cohort of 795 selected patients from the Cardiovascular Hospital outside Heart Fuwai was established: 150 patients with severe coronary heart disease (sCHD) and 150 age-and sex-matched normal coronary angiographic con. Trols, ANC. We defined coronary artery angiography showing at least one main coronary artery lumen narrowing (> 80%) as the sCHD group and coronary artery lumen narrowing (< 20%) as the ANC group. METABOLOGICAL DETECTION METABOLOGICAL METABOLISM METABOLOGICAL ANALYSIS OF PLASMA FROM IONIZATION MODELS OF CATIONIC AND ANIONIC PHASES. The selected small molecular metabolites with definite qualitative information of secondary mass spectrometry were added to the multiple linear regression model (P 0.05) to analyze and correct the traditional risk factors of coronary heart disease (age, sex, body mass index, diabetes mellitus). Bonferroni correction (P8.2 x 10-5 (0.05/611)) was used to prevent class I errors caused by n tests, and logistic regression model was used to analyze the direction and course of the association between these small molecular metabolites and coronary heart disease risk. Results: Except for age and sex matching, the traditional risk factors of coronary heart disease in patients with severe coronary stenosis were significantly higher than those in the normal control group. A total of 105 metabolites (21 of which were unknown metabolites) were screened out after adjusting for the risk factors and batches of traditional coronary heart disease (P 0.05). There were significant differences in plasma levels between the two groups: 51 phospholipids, 10 carnitines, 8 free fatty acids, and 5 bile acid metabolites. Products, 3 amino acids and 7 undifferentiated metabolites (indole, pentoxylic acid, 4-pyridoxic acid, salicyluric acid, caffeine, testosterone, p-methoxyphenylacetic acid). The plasma metabolites (pyridoxic acid, palmitic acid, subunit) with the most significant difference were screened from 105 different metabolites by Bonferroni correction. Oleic acid, lipoacyl carnitine (14:1), cholic acid, phosphatidylglycerol (20:3/2:0), and these metabolites all showed significant positive correlation with the risk of coronary heart disease (odds ratio, OR value range 2.79-7.24). Conclusion: The metabolic fingerprints of CHD patients in China were obtained by non-targeted metabonomics method, and piperidine was reported for the first time. These three potential biomarkers, namely, oxalic acid, cholic acid and phosphatidylglycerol (20:3/2:0), are strongly associated with the pathophysiological process of coronary heart disease. These biomarkers provide a basis for exploring the pathogenesis of coronary atherosclerosis at the overall level and for seeking new therapeutic approaches. Pyridoxic acid has effects on vascular endothelial cell proliferation and inflammation of mononuclear macrophages. Impact Objectives: Our previous study identified a small molecule metabolite pyridoxine with significant differences between patients with severe coronary artery stenosis and normal controls on coronary angiography, and found that the risk of coronary heart disease increased by 4.879 times for each 1% increase in plasma pyridoxine. To investigate the role of pyridoxine in the development of atherosclerosis, we initially explored the effect of pyridoxine on two types of cells (human umbilical vein endothelial cells (HUVEC) and human monocyte line (THP-1) closely related to the pathological formation of coronary atherosclerosis at the cellular level. Human umbilical vein endothelial cells (HUVEC) and human monocyte lines (THP-1) were stimulated by pyridoxic acid at concentrations (20 nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L, 500 nmol/L, 1000 nmol/L, 2000 nmol/L) in vitro. The proliferation of human umbilical vein endothelial cells (HUVEC) and human monocyte lines (THP-1) at different stimulation points (12 h, 24 h, 48 h and 72 h) was analyzed by cell viability assay. Reproductive effects were assessed by real-time quantitative polymerase chain reaction (Real-time quantitative polymerase Chain ReactionqRT-PCR) with the expression of interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and cell adhesion factor (ICAM) at 48 h after pyridoxine stimulation. The proliferative activity of human umbilical vein endothelial cells (HUVECs) began to decrease markedly and decreased by 10.7% when the concentration of pyridoxine increased, and decreased by 2 000 nmol/L. After 72 hours of stimulation with pyridoxine, the proliferative activity of HUVECs decreased markedly from 20 nmol/L to 20 nmol/L, and with pyridoxine, the proliferative activity of HUVECs decreased significantly. The expression of IL-1beta, TNF-alpha and ICAM in THP-1 cells was significantly higher than that in blank control cells, and the expression of IL-1beta and ICAM in THP-1 cells was up to 1.5% when the concentration of pyridoxine was 1000 nmol/L and 2000 nmol/L. Conclusion: Pyridoxic acid can induce the proliferation inhibition of human umbilical vein endothelial cells, and the expression of human monocyte inflammatory factors (IL-1 beta, TNF-a, ICAM) is up-regulated. Therefore, it is speculated that the increase of plasma pyridoxic acid level and the formation and development of atherosclerosis. Long-term efficacy and safety of drug-eluting stents and bare metal stents in the treatment of multiple coronary artery disease Objective: Drug-eluting stents (DES) can effectively reduce the incidence of restenosis after metal stent implantation and the revascularization rate of target vessel or target lesion, but their coated drugs can lead to target vessel endothelial repair. Delayed and increased risk of fatal stent thrombosis has attracted attention and discussion in academia on the safety of DES use. Therefore, for patients with multiple coronary artery lesions, surgeons have begun to try the intervention strategy of drug-eluting stent and metal bare stent implantation (Hybrid stent), with a view to reducing drug-eluting stent implantation appropriately. The aim of this study was to evaluate the long-term efficacy and safety of a combination of drug-eluting stents (Hybrid stents) and bare metal stents in the treatment of multiple lesions of coronary artery disease. A total of 6495 patients with multiple coronary artery lesions underwent coronary interventional therapy (PCI) were divided into drug-eluting stent and bare metal stent implantation groups (Hybrid group, 848 cases) and drug-eluting stent implantation group (5647 cases). Clinical follow-up data included 30-day, 1-year and 2-year mortality, myocardial infarction, revascularization of target lesions, revascularization of target vessels, major adverse cardiac events and stent thrombosis. The cumulative incidence of various clinical events during the 2-year follow-up period was compared between the two groups, and the clinical events were analyzed by Cox proportional hazard model. Results: The COX proportional hazard model after propensity score matching showed target revascularization (risk ratio 2.38,95% CI 1.50-3.70), target revascularization (risk ratio 1.61,95% CI 1.15-2.27) and major adverse cardiac events (risk ratio 1.37,95% Cocoa) in the Hybrid stent group 2 years after surgery. The risk of all-cause mortality, myocardial infarction, and all-cause mortality / myocardial infarction was not significantly different between the two groups. The cumulative incidence of late thrombosis in the Hybrid stent group was significantly lower than that in the DES stent group at 2 years after surgery (P = 0.0112), while other stent thrombosis was cumulative in 2 years. Events: There was no significant difference in the incidence of positive thrombosis, positive + possible thrombosis, early thrombosis and very late thrombosis. Conclusion: For patients with multiple coronary lesions requiring multi-stent implantation, the long-term efficacy of drug-eluting stent implantation alone was significantly better than that of drug-eluting stent and bare metal stent implantation. The risk model and scoring system for perioperative myocardial infarction after percutaneous coronary intervention (PCI) Objective: There was no systematic and comprehensive study on the risk of perioperative myocardial infarction after PCI at home and abroad. This study was aimed at PMI formulated by American Society of Cardiovascular Intervention (SCAI). Definition, systematic screening of independent risk factors for perioperative myocardial infarction, and establishment of a PMI risk model and scoring system for a comprehensive assessment of the risk of perioperative myocardial infarction under the combined effects of multiple risk factors. A total of 3 371 patients (3 516 PCIs) were treated and monitored for perioperative cardiac injury markers. The patients were divided into perioperative myocardial infarction group and non-perioperative myocardial infarction group according to the occurrence of perioperative myocardial infarction (PMI) defined by SCAI. The independent risk factors of perioperative myocardial infarction were identified by logistic multivariate regression analysis and the perioperative myocardial infarction risk model was established. The predictive power and effectiveness of the risk model and scoring system were further validated by the Bootstrap method (sample size n = 3516, sample number B = 1000). Results: Of the 3 371 patients who had successfully undergone percutaneous coronary intervention, 107 were enrolled. Perioperative myocardial infarction occurred in 108 PCI patients (3.1%). The independent risk factors for perioperative myocardial infarction were age (OR) 1.037,95% confidence interval (CI): 1.016-1.058; P 0.01), multi-vessel therapy (OR: 1.697,95% CI: 1.095-2.629; P = 0.02). Treatment of fork lesions (OR 1.869, 95% CI: 1.213-2.878; P 0.01), total length of target lesions (OR 1.016, 95% CI: 1.009-1.024; P 0.01). The area under the ROC curve of the PMI scoring system was 0.71 (95% CI: 0.66-0.76) and H-L P = 0.5. The average area under the ROC curve of the PMI risk scoring system was 0.707 and the median ROC curve was obtained by internal verification of Bootstrap method. According to the quartile of PMI score, the first three quartile Q1-Q3 was defined as non-PMI high-risk group (corresponding risk score 10 points), while the fourth quartile Q4 was defined as PMI high-risk group (corresponding risk score < 10 points), and the perioperative risk of myocardial infarction was significantly higher in PMI high-risk group than in non-PMI high-risk group (P 0.01). Independent risk factors affecting perioperative myocardial infarction were systematically screened: age, total length of target lesion, multi-vessel therapy, to
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R541.4

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