【作者】 孙国林；

【导师】 肖颖彬；

【作者基本信息】 第三军医大学 ， 外科学， 2013， 博士

【摘要】 一、背景和目的我国有大量人口生活在高海拔缺氧地区，研究长期缺氧对机体的影响非常重要。法洛氏四联症由于心内异常的右向左分流,使得心肌和全身组织长期处于缺氧状态,引起一系列的病理生理改变,因而作为慢性缺氧模型得到了广泛研究。法洛氏四联症(Tetralogy of Fallot,TOF)是最常见的紫绀性先天性心脏病。典型的TOF包括室间隔缺损,右心室肥厚,右室流出道梗阻和主动脉骑跨。自从Lillehei及其同事在1954年第一次为一10岁病人成功施行根治术以来，这一最常见的紫绀性心脏病一直受到广泛的研究，是否一期根治术或二期手术仍有争论。手术年龄目前趋于低龄化。影响法洛四联症根治术效果的因素很多，术后患者病情复杂多变，短期内即出现明显的代谢紊乱，导致细胞及器官的功能障碍，是一个多因素、多环节、多途径的复杂过程，其病理生理机制尚未能得到准确而系统的阐述。体外循环(cardiopulmonary bypass,CPB)后继发出血仍是心内直视术后的严重并发症之一。CPB术中使用氨甲环酸(tranexamic acid，TA)可有效地减少术中和术后失血量，减少患者对血液制品的需求量，已得到众多学者的认同并在临床常规应用。TA的剂量和给药方式尚无统一定论。尽管TA不良反应少，也无研究表明不良反应随剂量增加而增加，但目前尚无该药的小儿药代动力学研究。代谢组学是近年迅速发展起来的新兴生命学科，它从代谢的角度研究生命的本质及疾病的发生、发展过程。基于核磁共振的代谢组学主要利用NMR波谱技术和模式识别方法，对生物体液或组织进行系统测量和分析，定量研究机体对病理生理刺激或遗传变异引起的代谢应答，对影响机体整体状况的多因素的研究对机体某一病理变化的全面情况进行探究。选择在全麻低温体外循环下行一期根治术的TOF病人，作为观察对象，应用模式识别与信号校正技术分析尿液一维~1H-NMR谱，研究婴幼儿法洛四联症患者手术前与CPB期间的尿液代谢组学特征,以尝试将基于NMR的代谢组学新技术用于研究个体差异显著的复杂先天性心脏病患者，探讨代谢组学方法在心脏围手术期的应用。并对小儿TA进行药代动力学研究。二、内容和方法1.经房-肺动脉施行施行婴幼儿法洛氏四联症根治术的临床研究方法：总结我院经房-肺动脉施行施行婴幼儿法洛氏四联症根治术的早期结果。2.婴幼儿TOF根治术体外循环期间氨甲环酸血液及尿药浓度及排泄率。方法：10例先天性TOF患儿，年龄(11±6.3)月，体重(7.36±2.08)kg，在开胸前应用TA100mg/kg，单次静脉缓慢注射(>10分钟)，CPB开始前再次注射100mg/kg。应用~1H NMR方法，检测不同时间段TA的血液及尿液浓度，并计算TA排泄率。3.长期缺氧对婴幼儿TOF尿液代谢组的影响。方法：TOF患儿10例为缺氧组，健康10例为对照组。采集禁食后清晨尿液约3ml，分别进行~1H NMR检测，采用模式识别分析方法处理数据。4.体外循环对婴幼儿TOF尿液液代谢组的影响。10例TOF手术患儿为实验组，术前为自身对照组。采集禁食后术晨尿液，以及10例TOF患儿CPB期间尿液约3ml，分别进行~1H NMR检测，采用模式识别分析方法处理数据。5.长期缺氧对婴幼儿TOF心肌代谢组的影响。方法：TOF患儿5例为实验组。同年龄的室间隔缺损并右室流出道狭窄的患儿7例为对照组。在心脏停跳、切开右室疏通流出道时,切除肥厚的肌束作为实验样本,主动脉阻断4-8分钟内取出右室流出道标本，液氮快速冷冻，NMR分析前-80oC保存。三、结果1.经房-肺动脉入路组（A组）无死亡，经右心室入路组（B组）住院期间2例死亡。1例死于严重的低心排并多脏器功能衰竭，1例死于恶性心律失常包括室颤和室扑。CPB时间(95.02±23.8分：85.23±22.63分, p=0.032),主动脉阻断时间(69.4±10.36分：61.17±9.38分, p=0.035),正性肌力药物应用时间(1.63±0.97天：2.1±1.09天, p=0.02),机械通气时间(26.62±12.48小时，33.02±17.55小时, p=0.033), ICU滞留时间(2.25±1.28天;2.85±1.46天, p=0.026)和心律失常发生率(3例,5.7%;10例,18.9%,p=0.038)，二组有显著性差异。RV/LV压力比值(0.45±0.13；0.41±0.1, p=0.091),住院时间(10.8±2.03天;10.6±2.37天, p=0.642),再次手术(3例,5.7%;0例, p=0.079)，新诊断的肾功能不全(1例,1.9%;2例,3.8%, p=0.558)，二组没有显著性差异。(表2-3)。A组中，2例婴儿因出血，一例因左肺动脉与跨瓣环补片处扭曲，再次开胸手术。2. CPB开始前(负荷剂量用药后约20min)、CPB开始后1h、手术结束时的血液TA浓度分别为(224.61±195.28)、(509.58±181.57)、(243.95±32.30)ug/ml。CPB开始前、CPB期间、关胸期间的尿液TA浓度(mg/ml,M±SD)，分别为4.28±1.12；3.7±2.86；4.45±2.65mg/ml。CPB开始前、CPB期间、关胸期间尿液TA量(mg, M±SD)，分别为199.7±142.1,341.6±302.3,400.1±357.0mg。至手术结束(用药后约3小时)TA总排泄量为57.48±19.66%。CPB开始前排泄率6.5±4.8mg/min，CPB期间排泄率4.5±4.9mg/min，关胸期间排泄率13.1±9.6mg/min，总排泄率6.2±3.4mg/min，各组均无显著性差异，p>0.05。TA在0.03～6.0mg/ml具有良好的线性关系(r=0.999)；平均回收率为99.61%，RSD为0.45％(n=6)。3.核磁共振氢谱的PLS-DA结果显示,从得分图中可以看出TOF与健康儿童两组尿液代谢组呈聚类型分布(图3-1),TOF病人尿液与健康儿童尿液能区分。TOF与健康儿童代谢物积分与内标DSS相对指数比较见表3-1，TOF病人尿液胆汁酸(δ0.623)升高(p=0.03)，二甲胺(δ2.76)，三甲胺(δ2.82)，马尿酸(δ7.82)均降低,(P分别为0.028,0.043,0.002)，有显著性差异。4.法洛四联症CPB期间与术前尿液代谢组学比较核磁共振氢谱的PLS-DA结果显示,从得分图中可以看出两组尿样代谢组呈聚类型分布(图5-2)。统计分析亮氨酸(δ0.93)，异亮氨酸(δ0.96,1.02)，缬氨酸(δ0.99,1.06)，异戊酸(δ1.13,1.35,1.60,2.03),谷氨酰胺(δ2.44)，柠檬酸(δ2.53)，琥珀酸(δ2.71),α-酮戊二酸(δ3.01),肌酐(δ3.04，4.04),3,4-去羟苯乙二醇(δ6.85),马尿酸(δ6.86，7.56，7.64，7.82),尼克酰胺(δ8.32)，甲酸(δ8.44)，CPB期间较术前下降,有显著性差异(P<0.05)。外源性甘露醇(δ3.67，3.77,3.80，3.88)CPB期间较术前升高，有显著性差异(P<0.05)。5. HR-MAS~1H NMR的PLS-DA结果显示,从得分图中可以看出两组心肌代谢组呈聚类型分布(图6-1)。TOF心肌与对照心肌中都出现乳酸峰，但无显著性差异；TOF心肌中甘油三酯-(CH2)-基团(δ1.31)的谱峰信号升高明显,含量高于对照组，有显著性差异。四、结论1.经房-肺动脉施行施行婴幼儿TOF根治术是可行的，对2岁以下的婴幼儿TOF效果满意。2.~1HNMR能够检测出TA的血液及尿液浓度。该TA用法血药浓度高，大于抑制有高度出血风险可能需要的血液浓度125ug/ml，提示可降低剂量。有约一半剂量在手术结束后排出体外。3.长期慢性缺氧影响婴幼儿肠道菌群的结构和活性，影响马尿酸的合成和排泄，影响胆红素的肠肝循环。4.婴幼儿TOF在CPB期间，肌肉分解增强，肠道菌群代谢受到进一步影响，肝脏肾脏功能出现损害，但基本能满足能量代谢需求。5.长期缺氧影响心肌有氧代谢，无氧酵解增强，但仍能维持静息状态下的能量需求。TOF心肌中甘油三酯-(CH2)-基团(δ1.31)的谱峰信号升高明显,可能意味着长期缺氧可能影响了在心肌细胞线粒体中进行的脂肪酸-氧化代谢的关键酶。更多还原

【Abstract】 Background:Tetralogy of Fallot (TOF) is the common cyanosis congenital heart disease. Rightventricular (RV) dysfunction is an important cause of morbidity and mortality after surgicalcorrection of TOF. Transatrial/transpulmonary repair avoids a ventriculotomy (in contrast tothe transventricular approach) aiming to preserve right ventricular structure and function.We performed a prospective randomised controlled trial in infancy with TOF undergoingthe primary repair.Tranexamic acid (TA) is an antifibrinolytic agent, which faces difficulties in theachievement and maintenance of a therapeutic concentration. Hence, it is required tomonitor the TA level in blood/plasma during the administration.Metabolomics is a powerful new technology that allows for the assessment of globalmetabolic profiles in easily accessible biofluids and biomarker discovery in order todistinguish between diseased and non-diseased status. We utilized this approach in a pilotstudy in urine and intact myocardium samples from TOF patients and controls. Todistinguish TOF patients from matched controls with the global metabolic profiling andsubsequent multivariate analysis. To explore the urine metabonomic profile of TOF patientsbefore and during cardiopulmonary bypass.To detect specimens of hypertensive myocardium with high resolution magic anglespinning1-Hydrogen Nuclear Magnetic Resonance spectroscopy(HR-MAS~1H-NMRS)，toanalyze their soluble metabolites，discuss its metabolism response to chronic hypoxia inmyocardium．1. Primary repair of Tetralogy of Fallot in infants: transatrial/transpulmonary ortransventricular approach.A prospective controlled clinical trial was conducted in infancy with TOF undergoingthe primary repair. One-hundred and six patients recruited were divided into transatrial-transpulmonary approach group (Group A)(n=53) and transventricular approachgroup (Group B)(n=53) depending on the different surgical technique.2. The plasma and urine concentration of tranexamic acid duringcardiopulmonary bypass in infants10patients with Tetralogy of Fallot received an initial dose100mg/kg of TA givenover10min followed by an infusion of100mg/kg before the initiation of cardiopulmonarybypass (CPB). Plasma and urine TA concentrations were detected by~1H NMR.3. Study on Urine Metabolomics of Tetralogy of Fallot Infants using Hydro-Nuclear Magnetic Resonance SpectrometerTotal10infants with Tetralogy of Fallot (TOF) and10healthy infants were enrolled inthis study and their urine metabolites were analyzed using Hydro-Nuclear MagneticResonance (~1H NMR) spectrometer for the Partial Least Squares Discriminant Analysis(PLS-DA).4. Study on Urine Metabolomics of Tetralogy of Fallot patients during theCardiopulmonary Bypass PeriodThe urine metabolites from total10TOF infants before and during thecardiopulmonary bypass period were detected by~1HNMR spectrometer analyzed for thePartial Least Squares Discriminant Analysis (PLS-DA).5.Intact myocardium metabolomics based on the HR-MAS~1H-NMRspectroscopy.There were total10infancy myocardiums from the right ventricular outflow tract withcyanotic (n=5) or acyanotic cardiac defects (n=7) were detected by HR-MAS~1H-NMRspectroscopy.Results1. Patient preoperative characteristics and procedure-related variables were similar.There was no death in Group A, while2patients died in Group B. There were significantdifferences in CPB time (95.02±23.8minutes versus85.23±22.63minutes, p=0.032),Cross-clamp time (69.4±10.36minutes versus61.17±9.38minutes, p=0.035), inotropicsupport (1.63±0.97days versus2.1±1.09days, p=0.02), intubation time (26.62±12.48hoursversus33.02±17.55hours, p=0.033), ICU stays (2.25±1.28days versus2.85±1.46days,p=0.026) and the incidence of arrhythmia (3,5.7%versus10,18.9%, p=0.038). No significant difference in RV/LV pressure ratio and hospital stay.2. Plasma TA concentrations were (224.61±195.28)ug/ml at20min after bolus,(509.58±181.57)ug/ml after60min on CPB,(243.95±32.30)ug/ml at the end of operation.Urine TA concentrations(mg/mL; M±SD) before CPB、during CPB、thoracic closing,4.28±1.12；3.7±2.86；4.45±2.65mg/ml respectively.Total urine TA dose(mg; M±SD)before CPB,during CPB, and thoracic closing,199.7±142.1,341.6±302.3,400.1±357.0mg/lrespectively.The total TA excreted (M±SD) is57.48±19.66%until operation finished about3hours.3. PLS-DA of urine~1H-NMR spectra revealed different metabolic spectra betweenTOF and the healthy control.The urine concentration of dimethylmine(δ2.76), trimethylmine(δ2.82)mandhippurate(δ7.82) is decreased significantly in TOF group compared with the control,(p=0.028,0.043,0.002, respectively), the urine concentration of bile acid(δ0.623) isencreased significantly in TOF group compared with the control,(p=0.03).4. PLS-DA of urine~1H-NMR spectra revealed different metabolic spectra betweenbefore and during CPB, demonstrated that the metabolic characteristics of the two groupswere significantly different. Compared with before operation, Leucine(δ0.93)，isoleucine(δ0.96,1.02)， valine(δ0.99,1.06), isovalerate(δ1.13,1.35,1.60,2.03),glutamine(δ2.44),Citrate(δ2.53)， succinic acid(δ2.71), α-ketoglutarate(δ3.01), creatine(δ3.04，4.04),Hippurate(δ7.82), Nicotinamide (δ8.32)，Formic acid(δ8.44)，decreased significantly duringCPB, while exogenous Mannitol(δ3.67)encreased significantly.5. PLS-DA of myocardium NR-MAS~1H-NMR spectra revealed different metabolicspectra between TOF and control, demonstrated that the metabolic characteristics of the twogroups were significantly different. Only the triglyceride(δ1.31) is higher significant in theTOF myocardium than that of control.Conclusion1. Transatrial/transpulmonary repair of TOF is associated with excellent surgicalresults and immediately follow-up.2. A100mg/kg initial dose of TA followed by an infusionof100mg/kg before theinitiation of CPB is sufficient enough to provide an effective plasma concentration125ug/ml for the bleeding patients with high risk. The dose of TA could be decreased. About50%of total dose excreted off in3hours.3. Chronic Hypoxia alters metabolism of intestinal flora in infants.4．Characteristic metabolic products in two groups can be identified by~1H-NMR basedmetabonomics analysis. Metabonomic study is a feasible and promising way to detect thevariation of urine metabonomics of complex open heart operation patients.5. Chronic hypoxia effect the energy metabolism of myocardium and the key enzymeof fatty acid oxidative metabolism in the myocardial mitochondria.These findings highlight the potential of metabonomics as a novel approach forfundamental investigations of hypoxia interactions as well as for disease surveillance andcontrol. It holds promise for development of novel diagnostic approaches at individual andpopulation levels. Future studies should look at extending this work to other aged patientsmodels. We believe that metabonomics also represents an ‘omics’ science that now requirescomprehensive assessment and validation for its potential in individual and communitydiagnosis, particularly with regard to responses to treatment.更多还原