【作者】 王小华；

【导师】 龙村；

【作者基本信息】 北京协和医学院 ， 麻醉学， 2012， 博士

【摘要】 研究总体思路：深低温停循环是主动脉手术及复杂先心病手术中常用的体外循环方式,通过降低温度同时停循环创造无血、安静的手术视野。但是深低温停循环后的脑损伤无法避免。本研究从深低温停循环的脑损伤的临床监测及预防、深低温停循环脑损伤的机制、深低温停循环脑损伤的有效标记物三个方面,总体而全面和探讨深低温停循环后的脑损伤和脑保护。为深低温停循环脑保护提供全面的策略,也为深低温停循环脑保护提供全新的心路和方向。本研究第一部分首先通过深低温停循环选择性脑灌注下NIRS和TCD监测,探讨深低温停循环条什下最合适的选择性脑灌注流量,通过对流量与脑氧饱和度的相关性分析,以脑氧饱和度变化为参考,为临床上选择合适的脑灌注流量,并最大限度的减少深低温循环对大脑血供及氧供的影响提供理论依据。通过第一部分的TCD和NIRS的研究发现,与深低温停循环前相比,大脑中动脉流速、脑氧饱和度和静脉氧饱和度都显著降低,提示在深低温停循环选择性脑灌注期间的确存在不同程度的脑部的缺血缺氧。在主动脉阻断后的大脑中动脉的血流频谱中，能清晰的看到微栓信号。进一步提示我们,在深低温停循环期间有微栓进入大脑,并对脑功能产生影响。提示我们进一步研究深低温停循环的病理生理机制,为深低温停循环后脑损伤的治疗提供有效的保障。本研究的第二部分,在第一部分对深低温停循环期间经颅多普勒和近红外光谱脑氧饱和度开展临床监测的基础上,确实发现深低温停循环中的脑血流减低的现象和深低温停循环期间脑栓子的出现；由于各种物理化学的损伤最终导致的脑内保护性蛋白和损伤性蛋白的表达失衡,而蛋白的转录后调控机制是蛋白表达的中心环节,且相关研究尚未有报道,因此研究者在第二部分中设计通过建立新生猪的深低温停循环模型,深入探讨深低温停循环后的重要蛋白转录后调控机制,即多物种中广泛存在的microRNA的调控机制,通过转录后水平的调控机制的研究,深入到分子和基因水平探讨深低温停循环的脑损伤机制,为临床上深低温停循环后病人的不同的脑损伤情况的,为揭示深低温停循环多因素作用的分子生物学本质,为深低温停循环的脑损伤机制的研究开辟新的思路。通过深低温停循环microRNA差异性表达的研究,我们发现了包括miR-194、miR-200c、miR-122、miR-10b等发现35个差异性表达的microRNAs,并通过生物信息学分析,发现这些microRNA参与多种脑损伤相关性疾病,及信号转导通路。本研究的第三部分,在前期研究的基础上,利用动物模型芯片检测的结果,对深低温停循环病人的血浆脑损伤相关的microRNA标记物进行qRT-PCR分析,通过对深低温停循环后不同时间点的脑功能评分,对miR-194、miR-200c、miR-122、miR-10h的差异性表达及CNS评分的相关性分析,发现miR-194与患者深低温停循环术后的脑功能状况具有良好的相关性。总之,对深低温停循环合适流量的选择、大脑功能的监测的改进,及对深低温停循环机制的探索,甚至是未来在深低温停循环脑组织microRNA研究的基础上,其他药物和非药物治疗干预的研发和应用,都将为经历深低温停循环下,复杂血管手术、复杂先心病血管手术病人带来福音。第一部分深低温停循环选择性脑灌注条件下联合应用TCD和NIRS监测大脑功能的研究背景与目的本研究的目的是联合应用经颅脑血流多普勒(transcranial cerebral Doppler, TCD)和近红外光谱脑氧饱和度监测仪(near-infrared spectroscopy cerebral oxygen saturation, NIRS)监测深低温停循环选择性脑灌注条件下,大脑中动脉血流流速(The middle cerebral artery mean veloci ty, VmMCA)和局部脑氧饱和度(regional cerebral oximetry, rSO2)的改变,为降低深低温停循环期间脑损伤的保护策略提供临床依据。方法择期行深低温停循环选择性I脑灌注下行主动脉弓重建术的12个病人,麻醉诱导后将经颅脑血流多勒的探头放置于颞窗,近红外光谱脑氧饱和度监测仪探头放置于病人的前额来收集术前、术中和术后的血流动力学参数和大脑中动脉血流流速及局部脑氧饱和度。术中深低温停循环联合顺行选择性脑灌(antegrade selective cerebral perfusion, ASCP)流率5-10ml·kg(-1)·min(-1)。应用线性回归模型对平均动脉压(MAP)、泵流量、大脑中动脉血流流速(VmMCA),和混合静脉氧饱和度(SVO2)进行相关性分析。结果术中监测发现平均动脉压(Mean arterial pressure, MAP)和脑氧饱和度(rSO2)之间没有显著的相关性。大脑中动脉平均血流流速和脑氧饱和度有显著相关性,并且主泵流速与脑氧饱和度之间也有显著相关性。于深低温停循环选择性脑灌注之前相比,大脑中动脉流速,脑氧饱和度和静脉氧饱和度都显著的降低。但大脑中动脉血流流速和脑氧饱和度在术后回到体外循环前的水平。在停机后,深低温停循环选择性脑灌注的流量与大脑中动脉流速和脑氧饱和度表现出明显的正相关。在深低温停循环期间,当选择性脑灌注流量低于10ml/kg/min时,TCD无法监测到大脑中动脉(Cerebral middle arterial,MCA)血流信号。当选择性脑灌注的流量高于10ml/kg/min时,大脑中动脉的血流流速维持在正常水平而脑氧饱和度大于45%。结论联合应用经颅脑血流多普勒和近红外光谱脑氧饱和度监测仪能够有效的监测深低温停循环选择性脑灌注条件下的大脑功能,并为降低深低温停循环选择性脑灌注条件下降低脑损伤提供指导。第二部分深低温停循环脑损伤条件下海马区差异性miRNAs表达变化及其机制探讨背景与目的深低温停循环(Deep hypothermic circulatory arrest,DHCA)是复杂先心病心外科手术期间应用的一项重要技术。先天性心脏病是最常见的出生缺陷,由于灌注技术、药物、围术期监护的改进,使得经历极为复杂的手术操作的患儿的死亡率下降,先心病外科校正术后的存活率的增加,使人们把目光聚焦到长期预后,特别是神经发育的进展和患儿生活质量的改善。对经历心外科手术的患儿的长期随访发现,这些患儿在神经发育功能不良具有特征性的表现,而深低温停循环后脑损伤是复杂心脏校正术后最重要的并发症。常见的并发症包括：惊厥、舞蹈症、认知功能损伤、执行功能缺陷、语言功能障碍、视觉空间和视觉运动技巧障碍、注意力异常、行动延迟、学习障碍。对于深低温停循环相关的脑损伤处理及损伤预后的干预关键是对于深低温停循环脑损伤神经病理过程及其机制的理解。然而深低温停循环后脑损伤的确切的分子机制目前尚不清楚。近期深低温停循环后脑损伤的基因及蛋白水平的研究日渐增多,但转录后水平的调控尚未有相关的报道。microRNAs是18~22nt的小分子RNA,在体内参与多种生理病理机制的调控,是一种高效低耗的调节方式,最近研究提示microRNAs涉及各种脑损伤的发生及进展。本研究利用miRNAs基因芯片技术检测深低温停循环小猪脑损伤模型中的miRNAs表达变化,探讨深低温停循环后脑损伤miRNAs转录后调节机制,为深低温停循环后的脑损伤的预防和治疗开辟一个新的思路。方法新生猪(体重2.0-2.5kg)构建深低温停循环模型,深低温停循环组3只,假手术组3只。取海马组织提取总RNA,进行Affimatrix miRNAs多物种芯片杂交,后行qRT-PCR验证,生物信息学分析来分析一系列差异性表达的miRNAs涉及的脑部疾病。预测特异性miRNAs的潜在靶点,后通过MAS分析系统对预测靶点进行G0功能分析及KEGG探讨涉及的信号通路分析,探讨在转录后水平差异性表达的microRNA可能参与的调节机制。结果深低温停循环组海马区有35个miRNAs与假手术组海马区比较有表达改变。13个miRNAs显著上调(包括miR-23a,miR-27a,miR-182等),22个miRNAs显著下调(包括miR-10b,miR-200c,miR-210,mIR-150,mi R-194,miR-122等)。对这些miRNAs靶点预测的生物信息学分析揭示多个靶点和生物功能涉及DHCA的病理生理过程,其中包括信号转导,转录调节,凋亡和炎症反应。结论DHCA后脑损伤涉及一系列miRNAs的表达改变,为深低温停循环脑损伤的机制研究提供了新的理论依据和研究思路。第三部分深低温停循环条件下血浆中脑特异性microRNA表达与脑损伤的相关性研究背景与目的深低温停循环后脑损伤神经功能损伤情况的诊断及后期神经功能恢复情况的判断是心外科术后心外科医师及体外循环灌注师共同面临的挑战。开发新的特异性的诊断标记物是改进诊断、改善预后的关键环节。脑损伤后血液学检测是一种能够相对容易的获得的生化测定来源。本研究在前期深低温停循环动物实验研究的基础上,测定血液中脑损伤相关特异性microRNA的表达改变,并同时对患者的神经系统损伤状态进行国际公认的神经计量评分,从而找出与脑损伤密切相关的特异性microRNA,为脑损伤的诊断和评定提供理论依据。方法在伦理委员会批准的前提下,临床上随机选择21例主动脉夹层患者,拟行深低温停循环顺行性选择性脑灌注条件下全弓置换术。按照加拿大神经量表(CANADIAN NEUROLOGICAL SCALE, CNS)对术后不同时间点病人的神经状况进行评分。保存深低温停循环前及术后不同时间点的患者血液,提取总RNA。设计miR-194、miR-200c、miR-122、miR-10b特异性的茎环引物。实时荧光定量PCR的方法检测不同时点血浆样本4种microRNA的表达差异。最后,对差异性表达的miRNAs与CNS评分进行相关性分析。结果深低温停循环后病人血浆中miR-194的表达在术后0h,术后12h,术后18h,术后24h,术后36h时间点与术前相比明显降低(P=0.000),深低温停循环术后12小时比术后18h时点的miR-194表达显著较低(P=0.012),深低温停循环术后12小时比术后18h时点的miR-194表达显著较低(P=0.000)。深低温停循环后,miR-194在停循环术后各个时点都显著性降低,术后12h,病人血浆中的miR-194水平达到最低。miR-194的表达2-△CT(×103)值术前1.2629±0.3622。miR-194的表达2-△CT(×103)值术后0.6699±0.3896。术前与术后有显著的统计学差异(P=0.005)。结果显示深低温停循环前及深低温停循环后各个时间点,血液中miR-194的表达改变与深低温停循环前后的加拿大神经量表评分成正相关(r=0.574,P=0.000),拟合优度检验R2=0.33。miR-200c、miR-10b、miR-122与CNS评分没有显著相关性。结论深低温停循环前及深低温停循环后各个时间点,血液中miR-194的表达改变与深低温停循环前后的神经功能评分成正相关,反应血液中miR-194的表达改变与深低温停循环脑神经功能异常相关,提示miR-194能够作为神经功能损伤的标记物。后续还需对miR-194参与的功能进行进一步研究。更多还原

【Abstract】 General design of the study Deep hypothermic circulatory arrest commonly used in aortic surgery and complex congenital heart disease correction surgery. Deep hypothermic circulatory arrest can create no blood and quiet operative field by lowering the temperature at the same time stop the circulation.Brain injury after deep hypothermic circulatory arrest can not be avoided. In this study, we design three aspects to discuss deep hypothermic circulatory arrest brain damage and brain protection comprehensively. Provide a comprehensive strategy for deep hypothermic circulatory arrest and cerebral injury and a new direction and ideas for cerebral protection.The first part of the study is NIRS and TCD monitoring for deep hypothermic circulatory arrest.The purpose of this study is through the analysis of VmMCA flow and cerebral oxygen saturation to explore most suitable selective cerebral perfusion flow rate for DHCA and ASCP.Use cerebral oxygen saturation as a reference for clinical brain perfusion flow rate, to minimize the impact for deep hypothermic circulatory arrest on cerebral blood supply and oxygen to provide a theoretical basis. In this study we find thatthe middle cerebral artery flow velocity and cerebral oxygen saturation and venous oxygen saturation was significantly lower compared with that indes before deep hypothermic circulatory arrest.Flow spectrum of the middle cerebral artery after aortic cross-clamping can clearly see microthrombus signal.that may suggests that micro-bolt into the brain during deep hypothermic circulatory arrest and brain function maybe have some influence. That result prompted us to further study for study the pathophysiology of deep hypothermic circulatory arrest, finally to provide effective protection of the cerebral injury.The second part of this study, on the basis of preliminary studies,we establish piglet deep hypothermia circulatory arrest model to explore the post-transcriptional regulation mechanism of deep hypothermic circulatory arrest in-depth molecular and genetic level.By microarray technique, we explode dyregulatied23microRNAs, that including of miR-194, miR-200c. miR-122et al. And bioinformatic analysis found that those microRNAs involved in a variety of brain injury related diseases, and the signal transduction pathway.The third part of this study, on the basis of preliminary studies using animal models of DHCA, and basic on the microarray results, we collected patient’s blood who undergoing DHCA, detect the different expression of these microRNA before and after DHCA at different time points. Meanwhile, the expression level of microRNA and CNS score correlation analysis reflected that miR-194in patients with deep hypothermic circulation and brain functional status has a good correlation.All in all, The choice of the appropriate flow rate and the monitoring of brain function improvement during deep hypothermic circulation arrest, and the exploration of deep hypothermic circulatory arrest mechanism, even on the basis of brain tissue microRNA research of deep hypothermic circulatory arrest, other drugs and non-drug application of therapeutic interventions will help the development cerebral protection of DHCA. All these delevlopment will bring the patient of the complexity of vascular disease and complex congenital heart disease patients the Gospel. Part I Real-Time Continuous Neuromonitoring Combines Transcranial Cerebral Doppler with Near-Infrared Spectroscopy cerebral oxygen saturation during deep hypothermic circulatory arrestObjective The purpose of this investigation was to use combined transcranial cerebral doppler (TCD) and near-infrared spectroscopy cerebral oxygen saturation (NIRS) during total aortic arch replacement (TAAR) to monitor middle cerebral artery blood flow velocity and regional cerebral oximetry (rSO2) changes to provide a clinical basis for protective measures that may decrease injury of the central nervous system.Methods Consecutive12adult patients underwent deep hypothermic circulatory arrest (DHCA) and antegrade selective cerebral perfusion (ASCP) during TAAR. A TCD probe was placed at the temporal windows after induction of anesthesia and the NIRS probe placed on the forehead of patients to collect preioperative. intraoperative. and postoperative hemodynamic parameters, and cerebral blood flow (CBF) and rSO2during cardiopulmonary bypass (CPB).Result In this retrospective case series, all patients survived, and there were no postoperative neurologic complications. There was no significant correlation between the mean arterial pressure and rSO2. The middle cerebral artery mean velocity (VmMCA) and rSO2were significantly correlated, and main pump flow significantly correlated with rSO2. After ASCP, VmMCA, rSO2, and venous oxygen saturation were significantly lower than before ASCP, but VmMCA and rSO2returned to pre-CPB levels postoperatively. After off pump, the flow of ASCP showed a significant positive correlation with VmMCA and rSO2. During DHCA when ASCP flow was lower than5ml/kg/min, TCD could not detect the MCA. Blood flow signal. When the flow of ASCP was above keeping around10ml/kg/min, MCA CBF velocity was maintained and rSO2>45%.Conclusion The combination of TCD and NIRS can be effective in monitoring brain function during DHCA with ASCP and may provide a guide for decreasing brain injury during the TAAR procedure. Part Ⅱ MicroRNAs Expression Profiling of Cerebral Injury in the Piglet Model of Cardiopulmonary Bypass Undergoing Deep Hypothermic Circulatory Arrest.Objective The cerebral injury after DHCA is serious problem and little is known about this molecular mechanism. In this study, we constructed DHCA piglet model to investigate expression profile of miRNAs associated with DHCA brain injuryMaterials and Methods DHCA model was conducted by six male piglets (2.0-2.5kg) including three from the DHCA group and three from the Sham group, hippocampus were harvested for miRNA microarray analysis. All microarray data were analyzed by Significance Analysis of Microarrays (SAM), and this result was further confirmed by qRT-PCR assay, biological information analysis to predict the target of the panels of miRNAs, then the GO analysis to find the function of the target by MAS.Results Thirty-five miRNAs were differentially expressed in hippocampus after DHCA procedure. Thirteen miRNAs (miR-23a, miR-27a. miR-182, et al) were significantly up-regulated and twenty-two miRNAs (miR-10b, miR-200c, miR-210, miR-150, et al) were down-regulated in DHCA hippocampus. Bioinformatic analysis of the predicted targets for this panel of miRNAs revealed multiple protein targets, biological processes and functions involved in pathphysiology of DHCA cerebral injury including signal transduction, transcriptional regulation, apoptosis, and inflammatory.Conclusion DHCA cerebral injury involves a set of miRNAs dysregulated. miR-10b, miR-200c, miR-210, miR-150, miR-23a, et al play important role in regulate cerebral injury. Part Ⅲ Expression profile of brain microRNAs in circulation is the sensitive biomarker of brain injury during deep hypothermic circulatory arrestObjective Diagnosis of the brain injury, detection of neuropsychological changes, and the judgment of neurological function are the common challenges of cardiac surgeon and perfusionists. The development of new specific biomarker is the key part to improve diagnosis and prognosis of deep hypothermic circulatory arrest brain injury. Hematology testing is a relatively easy access to biochemical measurement. In this study, basic on our previous microarray result about deep hypothermic circulatory arrest in piglet module. We choose significantly express microRNA to detect the expression change among the different time of DHCA. We purpose of this study is identify specific microRNA which is closely related to the brain injury and provide a theoretical basis for assessment of neurological injury.Materials and Methods Subject to the approval of the Ethics Committee, we selected21patients with aortic dissection randomly with proposed total arch replacement during deep hypothermic circulatory arrest and antegrade selective cerebral perfusion. Neurological conditions of patients with preoperative and postoperative different time points scored in accordance with the Canadian Neurological Scale (CANADIAN NEUROLOGICAL SCALE, CNS). We saved the blood of patients at different time points, extracted total RNA, designed miR-194, miR-200c, miR-122, miR-10b-specific stem-loop primers. Real-time quantitative PCR method to detect different point of blood samples of four kinds of microRNA expression differences. Finally, analysis correlation of differentially expressed miRNAs and CNS score.Results Deep hypothermic circulatory arrest, miR-194expression of the patient’s blood at postoperative0h,12h,18h,24h, and36h time points compared with preoperative decreased significantly (P=0.000). MiR-194expression2-△CT (×103) value of preoperative is1.2629±.3622. MiR-194expression2-△CT (×103) value of postoperative is0.6699±3896. Before and after surgery have a statistically significant difference (P=0.005). After deep hypothermic circulatory arrest, miR-194level at postoperative12h is minimum. The results also showed that the Canadian Neurological Scale scores with miR-194’s expression changes in blood with deep hypothermic circulatory arrest was positively related (r=0.574, P=0.000), to be co-goodness test R2=0.33. But miR-200c, miR-10b, miR-122and CNS score no significant correlation.Conclusion pre-and post-Deep hypothermic circulatory arrest the expression changes of miR-194and CNS score a positive correlation, the reflected that expression changes of miR-194in the blood correlated with neurological dysfunction, suggesting that miR-194can be used as biomarkers of neurological deficit. Follow-up study will focus on the function of miR-194participate in future.更多还原