NOS在颅内动脉瘤发病机制中的作用及颅内多发动脉瘤个体化治疗研究

发布时间：2018-05-20 14:27

本文选题：颅内动脉瘤 + 病理学　；参考：《山东大学》2016年博士论文

【摘要】：第一部分 NOS在颅内动脉瘤发病机制中的作用研究研究背景：颅内动脉瘤(Intracranial Aneurysm, IA)是动脉管壁病理性局限性扩张产生的脑血管瘤样突起,其破裂是引起人类自发性蛛网膜下腔出血(Subarachnoid Hemorrhage, SAH)最常见的原因。IA的发病率在我国脑血管意外中排第三位,仅次于缺血性脑卒中和高血压脑出血。该病诊治的难点在于临床症状隐匿,大多数在破裂后才被发现,致死率和致残率极高。目前对于IA无理想的早期预防和筛查手段,是由于该病的病理学改变及发病机制尚未完全明确。最近的研究表明,受损动脉壁在血流动力学改变与炎性反应的共同作用下暴露出一些抗原分子,引发分子水平上的一系列的级联反应,最终导致动脉壁降解,引起IA的形成及破裂。其中一氧化氮合酶(Nitric Oxide Synthase, NOS)在这一分子级联反应中发挥重要作用。目前已有动物实验表明IA模型中存在特征性的病理学及NOS表达水平的改变,由于人体IA标本难于获取,尚缺乏活体动脉瘤标本的病理学及NOS表达水平研究。如果能发现人体动脉瘤标本中NOS明显改变,则可以证实NOS可能在IA形成过程中发挥重要作用,将对IA的早期预防和筛查具有重要意义。众多的研究表明,在IA形成和破裂过程中,除了NOS外尚有大量的细胞因子及炎性介质等蛋白分子的表达及活性均会发生改变。因此,在这些纷繁复杂的细胞因子和蛋白分子相互作用网络中,筛选出一些相对重要的枢纽环节或关键因子,明确它们在IA病理生理中的调控作用,就可能成为IA发病机制研究的突破口。以同位素标记相对和绝对定量(Isobaric Tags for Relative and Absolute Quantitation, iTRAQ)技术为基础的定量蛋白质组学方法是近年来发展起来的一种高通量蛋白分子筛选方法,是目前差异表达蛋白分子筛选最强有力的工具。本研究拟对手术获取的脑动脉瘤标本进行常规病理检查,观察动脉瘤壁的病理学变化及超微结构改变；通过免疫组化方法检测NOS在动脉瘤标本内表达情况；同时通过iTRAQ技术筛选动脉瘤标本中差异表达蛋白分子,探讨它们在动脉瘤形成和破裂中可能的作用,为进一步阐明动脉瘤发病分子机制提供依据,同时也为动脉瘤生物学治疗的发展奠定基础。目的：观察动脉瘤的病理学及超微结构改变,检测NOS在颅内动脉瘤中的表达,通过iTRAQ技术筛选动脉瘤差异表达的蛋白分子,探讨它们在动脉瘤形成和破裂中可能发挥的作用,为进一步阐明动脉瘤发病分子机制提供依据。方法：选取山东大学齐鲁医院及潍坊市人民医院神经外科2012年5月～2014年11月神经外科手术夹闭的脑动脉瘤标本,同时以颅脑外伤手术患者头皮断裂的颞浅动脉(Superficial Temporal Artery, STA)及皮层动脉作为对照,1.用常规光镜、电镜观察IA及正常血管的病理形态学变化及超微结构改变。2.免疫组化SABC法测定动脉瘤中诱导型一氧化氮合酶(Inducible Nitric Oxide Synthase, iNOS)表达水平,同时测定动脉瘤标本中NOS蛋白含量。3.iTRAQ技术筛选颅内动脉瘤标本中差异蛋白分子。结果：1.动脉瘤的病理变化：HE结果显示动脉瘤典型的三层结构消失,内膜连续性中断,内弹力板较薄,消失或中断,内皮细胞变性或坏死。中膜平滑肌细胞少见,细胞外基质破坏严重,并见大量炎性细胞浸润,其中以巨噬和中性粒细胞为主。电镜超微结构观察显示动脉瘤内皮细胞损伤、坏死,可见细胞固缩或空泡变性；中膜正常内部结构消失,细胞外基质模糊不清,平滑肌细胞坏死,部分见凋亡小体；瘤壁可见炎性细胞浸润,主要为巨噬细胞。而对照组动脉的结构是正常的,内皮细胞完整、均匀,中膜内平滑肌纤维排列有序、密集,基质纤维清晰可见,并没有发现炎症反应。2.NOS表达：iNOS在25例对照组标本中23例无表达(23/25),2例弱表达(2/25),36例动脉瘤组中6例中表达(6/36),30例强表达(30/36),动脉瘤组iNOS表达水平与对照组相比显著增加,差异有统计学意义(P0.05)。动脉瘤组iNOS含量为13.3129±2.4581 U/mg蛋白,对照组为7.5623±2.0321 U/mg蛋白,iNOS在动脉瘤组明显增高,有统计学意义(P=0.0320.05)。动脉瘤组总一氧化氮合酶(Total NOS)为24.5631±6.5684U/mg蛋白,对照组为22.1325±6.1358 U/mg蛋白,差异无统计学意义(P=0.5620.05)。动脉瘤组中iNOS/TNOS之比明显升高,差异有统计学意义(P=0.0390.05)。3.iTRAQ结果：通过iTRAQ技术共鉴定出有定量信息的蛋白分子816个,与对照组相比,差异2倍以上的有162个,包括80个上调蛋白和82个下调蛋白,其中与细胞外基质降解有关的基质金属蛋白酶-9(Matrix MetalloProteinase, MMP-9)上调3.7倍。上调蛋白主要是炎性反应相关蛋白,其次是细胞迁移、侵袭和细胞免疫补体复合物蛋白；除细胞骨架连接蛋白外,下调蛋白较多的是在内皮细胞迁移、增殖以及信号传导中发挥重要作用一类蛋白。结论：1.动脉瘤的病理及超微结构改变主要是内皮细胞坏死及炎症细胞浸润,特别是单核巨噬细胞,平滑肌细胞凋亡,细胞外基质破坏,提示内皮细胞坏死及炎症细胞特别是巨噬细胞浸润在动脉瘤壁病理改变中发挥重要作用。2.动脉瘤标本中以iNOS为主的NOS的表达增高说明,iNOS可能在动脉瘤发生发展过程中发挥重要作用。3.破裂动脉瘤有多种蛋白分子表达异常,它们可能参与了动脉瘤形成和破裂的分子机制。4.颅内动脉瘤的形成是由NOS介导的多种蛋白分子参与的复杂过程。第二部分颅内多发动脉瘤个体化治疗研究研究背景：颅内多发动脉瘤(Multiple Intracranial Aneurysms, MIAs)是指颅内同时存在2个或2个以上的动脉瘤。与单发性颅内动脉瘤(Single Intracranial Aneurysm, SIA)相比,破裂机会更多,诊治更加复杂,预后更差。MIAs发病率约占颅内动脉瘤的5%-30%,其检出率随着影像学尤其是3D-CTA、 3D-DSA的发展不断提高,处理MIAs将成为颅内动脉瘤治疗的重点。目前对于MIAs临床治疗措施,尚未达成一致意见,争议主要存在以下四个方面：一是MIAs中未破裂动脉瘤(Unruptured Intracranial Aneurysms, UIA)手术还是保守治疗；二是血管内治疗还是显微外科手术治疗；三是一期还是分期手术治疗；四是已发生脑血管痉挛的情况下手术时机的选择。颅内镜像动脉瘤(Mirror Aneurysms, MAs)是MIAs的一种特殊类型,指位于两侧对称位置的动脉瘤,由于其空间分布的特殊性,临床治疗较棘手。本文回顾性分析了我科收治经外科治疗的106例MIAs(包括37例MAs)患者的临床资料,多因素回归分析影响MIAs预后的临床因素,总结我科治疗经验,为MIAs个体化治疗策略的制定提供参考。目的：探讨MIAs及MAs的个体化治疗方案及MIAs患者的预后影响因素。方法：回顾分析2007年1月-2015年12月潍坊市人民医院神经外科收治的经外科手术治疗的106例MIAs(共232枚动脉瘤,其中包括37例MAs)患者的完整资料。根据患者具体情况,按照治疗措施分为三组,显微手术、介入栓塞、介入栓塞和显微手术联合治疗；按照治疗的时机分为仅处理责任动脉瘤,一期及分期处理动脉瘤三组。106例MIAs中开颅夹闭58例,介入栓塞44例,手术联合介入4例；其中39例仅处理责任动脉瘤,一期治疗53例,分期处理14例。37例MAs开颅夹闭21例,介入栓塞15例,手术联合介入1例；其中18例仅处理责任动脉瘤,一期治疗13例,分期处理6例。探讨的术前临床因素包括：患者性别、年龄、动脉瘤部位、侧别、数量、术前Fisher分级、Hunt-Hess分级、治疗措施、处理时机。出院时G08评分评估术后疗效。分别应用单因素分析及多因素Logistic回归分析术前各因素、治疗方案对治疗效果的影响。结果：出院时MIAs患者GOS评分5分75例,4分10例,3分9例,2分4例,1分8例。预后良好(4-5分)85例,差(1-3分)21例。MAs患者出院时GOS评分5分31例,4分2例,3分3例,2分0例,1分1例。预后良好(4-5分)33例,差(卜3分)4例。各治疗组GOS评分：介入44例中,良好37例,差7例；手术58例中,良好45例,差13例；介入+手术4例中,良好3例,差1例。MIAs患者年龄、部位、术前Fisher及Hunt-Hess分级经单因素分析显示与预后相关(P0.05),患者年龄越大,预后越差,动脉瘤位于后循环预后差,术前Fisher及Hunt-Hess分级级别越高,预后越差。多因素Logistic回归分析结果显示MIAs患者各因素中年龄、部位及Fisher分级与预后相关(P0.05)。结论：MIAsI临床多见,而MAs少见,二者均多见于后交通动脉,主要依靠DSA诊断,应在诊断全面的基础上,正确判断责任动脉瘤并早期积极治疗,对于非责任病灶需要综合评估出血风险,采取个体化治疗措施,尽量一期治疗所有动脉瘤,减少再出血风险。患者年龄、部位、术前Fisher和Hunt-Hess分级是影响MIAs患者术后疗效的关键因素。根据患者的实际情况选择开颅手术、介入栓塞或开颅手术+介入栓塞等个体化治疗方案是改善MIAs患者预后的关键。
[Abstract]:Part I research background of the role of NOS in the pathogenesis of intracranial aneurysm: Intracranial Aneurysm (IA) is the cerebral angiomatoid protuberance produced by the pathological localized dilatation of the arterial wall, and its rupture is the most common cause of human spontaneous subarachnoid hemorrhage (Subarachnoid Hemorrhage, SAH),.IA. The incidence of the disease is third in China's cerebrovascular accident, second only to ischemic stroke and hypertensive intracerebral hemorrhage. The difficulty in the diagnosis and treatment of the disease lies in the concealment of clinical symptoms, most of which are found after the rupture, and the mortality and disability rate are very high. At present, there is no ideal early prevention and screening for IA because of the pathological changes and hair of the disease. The mechanism of the disease is not completely clear. Recent studies have shown that the damaged arterial wall exposes some of the antigen molecules under the combination of hemodynamic and inflammatory reactions, triggering a series of cascade reactions on the molecular level, resulting in the degradation of the arterial wall and causing the formation and rupture of IA. Among them, the nitric oxide synthase (Nitric Oxide Synthas) E, NOS) plays an important role in the cascade reaction of this molecule. Now animal experiments have shown that there is a characteristic pathology and the change of NOS expression in the IA model. Because of the difficult to obtain in human IA specimens, the pathology of the specimens of living aneurysm and the level of NOS expression are still lacking. If NOS is found in the specimens of human aneurysm. The significant changes can be shown that NOS may play an important role in the formation of IA and will be of great significance for the early prevention and screening of IA. Many studies have shown that in the process of formation and rupture of IA, there are a large number of cytokines and inflammatory mediators, such as the expression and activity of protein molecules, except NOS. In the complex interaction network of cytokine and protein molecules, some important key links or key factors are screened, and their regulation role in the pathophysiology of IA may be a breakthrough point in the study of the pathogenesis of IA. Relative and absolute isotope labeling (Isobaric Tags for Relative and Absolute Q) Uantitation, iTRAQ) based quantitative proteomics is a high throughput protein molecular screening method developed in recent years. It is the most powerful tool for screening differentially expressed protein molecules at present. This study is to examine the pathological changes of the aneurysm specimens obtained by surgery to observe the pathological changes of the aneurysm wall. The expression of NOS in the aneurysm specimens was detected by immunohistochemical method, and the differentially expressed protein molecules in the aneurysm specimens were screened by iTRAQ technique, and the possible role of the proteins in the formation and rupture of aneurysms was explored to provide the basis for further elucidate the molecular mechanism of the aneurysm and also to act as a move. Objective: to establish the basis for the development of the biological treatment of the aneurysm. Objective: To observe the pathological and ultrastructural changes of the aneurysm, to detect the expression of NOS in intracranial aneurysms, to screen the protein molecules expressed differently by iTRAQ technique and to explore their possible role in the formation and rupture of aneurysms to further elucidate the incidence of aneurysms. The molecular mechanism provides the basis. Methods: the cerebral aneurysm of the Qilu Hospital in Shandong University and Department of Neurosurgery of the Shandong University from May 2012 to November 2014 was selected, and the superficial temporal artery (Superficial Temporal Artery, STA) and the cortical artery were used as the control, 1. Routine light microscopy and electron microscope observation of the pathological changes of IA and normal blood vessels and ultrastructural changes,.2. immunohistochemical SABC method was used to determine the expression of inducible nitric oxide synthase (Inducible Nitric Oxide Synthase, iNOS) in the aneurysm, and the determination of NOS protein content in the aneurysm specimens by.3.iTRAQ technique in the selection of intracranial aneurysm specimens Results: pathological changes of 1. aneurysms: HE results showed that the typical three layer structure of the aneurysm disappeared, the endometrium was interrupted, the internal elastic plate was thinner, disappearing or interrupted, the endothelial cells were denatured or necrotic. The middle membrane smooth muscle cells were rare, the extracellular matrix was destroyed seriously, and a large number of inflammatory cells were infiltrated. The ultrastructural observation showed that the endothelial cells of the aneurysm were damaged, necrosis, cell fixation or vacuolar degeneration; the normal internal structure of the middle membrane disappeared, the extracellular matrix was blurred, the smooth muscle cells were necrotic, and some of the apoptotic bodies were seen; the tumor wall showed inflammatory cell infiltration, mainly macrophages. And the control artery The structure was normal, the endothelial cells were complete and uniform. The smooth muscle fibers in the middle membrane were arranged in order, dense, and the matrix fibers were clearly visible. There was no expression of.2.NOS in the inflammatory reaction. 23 cases were not expressed (23/25), 2 cases of weak expression (2/25), 6 cases (6/36), 30 cases of strong expression (30/36), aneurysm and aneurysm in the 25 cases of control group. The expression level of group iNOS was significantly increased compared with the control group (P0.05). The iNOS content of the aneurysm group was 13.3129 + 2.4581 U/mg protein, the control group was 7.5623 + 2.0321 U/mg protein, iNOS was significantly increased in the aneurysm group, and was statistically significant (P=0.0320.05). The total nitric oxide synthase (Total NOS) was 24.5631 + 6.5684U (Total NOS) in the arteria group. /mg protein, the control group was 22.1325 + 6.1358 U/mg protein, the difference was not statistically significant (P=0.5620.05). The ratio of iNOS/TNOS in the aneurysm group was significantly higher, the difference was statistically significant (P=0.0390.05).3.iTRAQ results: 816 of the protein fractions with quantitative information were identified by iTRAQ technology, and 162 more than the control group, the difference was 2 times more than that of the control group. It includes 80 up-regulated proteins and 82 down regulated proteins, of which the matrix metalloproteinase -9 (Matrix MetalloProteinase, MMP-9), which is related to the degradation of extracellular matrix, is up to 3.7 times. The up-regulated protein is mainly inflammatory response related protein, followed by cell migration, invasion and cell immune complement complex protein, except for cytoskeleton connexin. More protein is a kind of protein which plays an important role in the migration, proliferation and signal transduction of endothelial cells. Conclusion: the pathological and ultrastructural changes of 1. aneurysms are mainly endothelial cell necrosis and inflammatory cell infiltration, especially mononuclear macrophages, smooth muscle cells apoptosis, extracellular matrix destruction, indicating endothelial cells necrosis and Inflammatory cells, especially macrophage infiltration, play an important role in the pathological changes of the aneurysm wall. The increased expression of iNOS based NOS in the.2. aneurysm specimens indicates that iNOS may play an important role in the development of aneurysm. There are various protein molecular tables in the aneurysm of.3. ruptured aneurysm, which may be involved in the formation of aneurysm. The formation of.4. intracranial aneurysms, the molecular mechanism of rupture, is a complex process involving multiple protein molecules mediated by NOS. The study background of the individualized treatment of second part intracranial multiple aneurysms: intracranial multiple aneurysms (Multiple Intracranial Aneurysms, MIAs) is the simultaneous existence of 2 or more than 2 aneurysms in the cranium. Compared with Single Intracranial Aneurysm (SIA), the chance of rupture is more, the diagnosis and treatment are more complicated and the prognosis is worse, the incidence of.MIAs is about 5%-30% of intracranial aneurysm. The detection rate is increasing with the imaging, especially the 3D-CTA, 3D-DSA, and the treatment MIAs will be the focal point of the treatment of intracranial aneurysm. Currently, the treatment of MIAs is the key to the treatment of the intracranial aneurysm. Bed treatment measures have not been agreed, the main four aspects of the dispute are the following four aspects: one is the unruptured aneurysm (Unruptured Intracranial Aneurysms, UIA) operation or conservative treatment; two is intravascular or microsurgical treatment; three is one stage or staging operation; four is cerebrovascular spasm. Mirror Aneurysms (MAs) is a special type of MIAs, which refers to the aneurysm located on both sides of the symmetrical position. The clinical treatment is difficult because of its spatial distribution, and the clinical treatment of 106 cases of MIAs (including 37 cases of MAs) in our department is reviewed. Bed data, multiple factor regression analysis of the clinical factors affecting the prognosis of MIAs, summarize the experience of our treatment and provide reference for the formulation of MIAs individualized treatment strategies. Objective: To explore the individualized treatment scheme of MIAs and MAs and the prognostic factors of MIAs patients. Methods: retrospective analysis of the nerve outside of Weifang People's Hospital in December January 2007. 106 cases of MIAs (a total of 232 aneurysms, including 37 cases of MAs) were treated with complete data. According to the specific conditions of the patients, the patients were divided into three groups according to the treatment measures, microsurgery, interventional embolization, interventional embolization and microsurgery combined treatment. Three.106 cases of aneurysm were treated with craniotomy in 58 cases, interventional embolization in 44 cases, and operation combined intervention in 4 cases, of which 39 cases treated only responsible aneurysm, one stage treatment 53 cases, 14 cases of.37 cases with 21 cases of MAs craniotomy, 15 cases involving interventional embolization, 1 cases with interventional embolization, and 18 cases only dealing with responsible aneurysm, one-stage treatment 13. 6 cases were treated. The preoperative clinical factors included: sex, age, aneurysm site, side, quantity, preoperative Fisher classification, Hunt-Hess classification, treatment measures, treatment timing. G08 score was used to evaluate the postoperative effect. The single factor analysis and multiple factor Logistic regression were used to analyze the preoperative factors respectively. Results: the GOS score of MIAs patients at discharge was 5 points, 4 points 10 cases, 3 points 9 cases, 2 points 4 cases, 1 8 cases, 8 cases with good prognosis (4-5 points), bad (1-3 scores) GOS scores in the patients at discharge. 37 cases were good, 7 cases were poor, 45 cases were good, 13 cases were bad, 3 cases were good in 4 cases of interventional + operation, and the age, location, Fisher and Hunt-Hess classification of.MIAs patients were correlated with prognosis (P0.05), the older the age was, the worse the prognosis, the poor prognosis of the aneurysm in the posterior circulation, Fisher and Hunt-Hess grading level before operation. The higher the prognosis, the worse the prognosis. The results of multiple factor Logistic regression analysis showed that the age, location and Fisher classification of the MIAs patients were associated with the prognosis (P0.05). Conclusion: MIAsI is more common, but MAs is rare, and all of the two are mostly found in the posterior communicating artery and mainly rely on the diagnosis of DSA. On the basis of a comprehensive diagnosis, the aneurysm should be correctly judged and early in the diagnosis of the responsible aneurysm. Active treatment requires a comprehensive assessment of the risk of bleeding for non responsible lesions, adopt individualized treatment measures, try to treat all aneurysms and reduce the risk of rebleeding. Age, location, preoperative Fisher and Hunt-Hess classification are the key factors affecting the postoperative effect of MIAs patients. Individualized treatment including embolization or craniotomy plus interventional embolization is the key to improve the prognosis of MIAs patients.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743

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