【作者】 张明铭；

【导师】 蒋宇钢；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 目的颅内动脉瘤在临床上以自发脑出血、脑血管痉挛、动眼神经麻痹等局灶症状为主要表现,是最常见的脑血管病之一。先天性动脉瘤占大部分人群,发病率达万分之一,以40-66岁最为常见。调查结果显示,约有10%的病人入院前死亡,但仍约有500万以上的隐匿性颅内动脉瘤未被发现和诊断。尽管在近十年里,动脉瘤的治疗取得了长足的进步,但其致死率仍高达30%-40%,且预后不甚理想。目前,该病发病机制仍存在广泛争议,致病机理尚不清楚。仅发现芬兰和日本人群中的发病率是其他人群的两倍,在遗传家系分析中找到了可能的相关基因,但并没有进行更深入的功能研究。近年来,研究者们用基因芯片来检测颅内动脉瘤相关基因mRNA水平的变化,发现Bcl-2,COL1A2,COL3A1,COL5A2,CXCL12,TIMP4,TNC这7个基因的表达水平在3个或以上研究中都发生了明显变化。这些在基因转录水平相关的研究均涉及到内皮细胞功能的紊乱,壁细胞的缺失,炎症细胞浸润以及细胞外基质的降解。小RNA (microRNA)是由18-25个核苷酸组成的单链非编码RNA。成熟小RNA通过与靶mRNA的3’UTR(非翻译区)互补结合在转录后水平调节其表达。据保守估计,小RNA调控约20%-30%的人类基因。研究显示,小RNA的异常调控是某些疾病发生的重要因素之一,如肿瘤和癌症。迄今为止,研究者们尚未明确任何颅内动脉瘤致病的信号通路和关键因子,也未能通过相关基因的动物模型模拟疾病的表型,更没有任何小RNA在颅内动脉瘤中的研究报导。我们长期从事颅内动脉瘤的临床工作,并在科研中积累了大量的实验成果。我们发现microRNA与颅内动脉瘤有着非常密切的关系。我们利用microRNA芯片对四例颅内动脉瘤样本进行小RNA表达谱分析,通过RT-PCR验证后得到18个异常表达的microRNA。同时利用mRNA芯片分析技术对两例颅内动脉瘤样本进行mRNA表达谱分析,通过数据筛选,得到11351个颅内动脉瘤mRNA表达量数据。利用IPA (Ingenuity pathway analysis)系统,建立了颅内动脉瘤发病相关通路的分子网络以及筛选这些miRNA对应的颅内动脉瘤发病相关的致病靶基因,发现这些异常表达的miRNA主要与血管内壁细胞凋亡、促进炎症细胞增殖与浸润、内皮细胞与平滑肌细胞和巨噬细胞的迁移等基因相关。本课题将进行miRNA及其靶基因在颅内动脉瘤致病机制的研究,探求其调控的分子网络在瘤体发生和发展中的作用,从时间和空间水平揭示miRNA调控颅内动脉瘤的分子体系。评估miRNA作为颅内动脉瘤生物标记的可能性,为颅内动脉瘤提供新的诊断位点和治疗干预新靶点。本课题将为颅内动脉瘤的分子机制提供有价值的研究成果。方法1.筛选在颅内动脉瘤中异常表达并具有显著生物学意义的microRNA。2.筛选在颅内动脉瘤中异常表达并具有显著生物学意义的mRNA。3.确定microRNA调控的关键靶基因在颅内动脉瘤组织中的表达水平,建立颅内动脉瘤发病的分子调控系统。4.分析颅内动脉瘤形成和发展过程中microRNA对细胞增殖、迁移和凋亡的影响,确定其在致病机理中发挥的功能。结果1.应用Agilent Human miRNA array AMADID1643芯片对4组动脉瘤样本及对照组织样本进行初步的筛选后,得到有异常表达的miRNA32种,对其中的29种miRNA进行RT-PCR验证(针对新采集的10组动脉瘤样本)后,有18种miRNA在颅内动脉瘤中异常表达。2.应用Agilent Whole Human Genome Oligo Microarray (4×44K)芯片对2组动脉瘤样本及对照组织样本进行筛选后,得到有异常表达的mRNA11351个。3.应用综合性分析系统IPA,对18种miRNA对应的靶基因进行功能分析,发现与mRNA芯片结果对应的靶基因对比有624个基因重叠。4.构建动脉瘤致病相关的分子网络后,异常表达的miRNA所对应的靶基因主要在以下方面参与颅内动脉瘤的致病：血管内皮细胞凋亡、炎症免疫细胞的增殖和浸润、内皮细胞和平滑肌细胞的迁移。5.分析IPA建立的动脉瘤致病机理的分子网络,炎症因子在颅内动脉瘤的形成和发展中扮演了重要角色。结论1. microRNA及其对应的靶基因在颅内动脉瘤的致病机理中扮演重要的角色。2. microRNA在颅内动脉瘤的形成中主要通过内皮细胞的凋亡、炎症免疫细胞的增殖和浸润、内皮细胞和平滑肌细胞的迁移等机制发挥作用。3.细胞免疫反应及细胞因子信号分子网络是颅内动脉瘤致病机理中最主要致病途径。更多还原

【Abstract】 BackgroundAs one of the most incident form of cerebrovascular diseases, intracranial aneurysm (IA) has a high mortality and undesirable prognosis with Spontaneous cerebral hemorrhage, cerebral vasospasm, oculomotor nerve palsy as the main clinical feature. Most of IA are congenital aneurysms, the incidence of IA is approximately1/10,000, a significant proportion of aneurysmal patients are of40-60age. MicroRNA are post-transcriptional regulators that bind to complementary sequences on target mRNA, roughly regulate about20%-30%human genes. Aberrant expression of micoRNA is involved in the pathogenesis of some cancer. The incidence in Finland and Japan is twice that of the rest of the population, However, large amounts of data created with each study, make a comparison or interpretation of results difficult in the genetic pedigree analysis.In recent years, researchers have used microarray gene expression studies on mRNA profiling of intracranial aneurysms. Seven genes (BCL2,COL1A2, COL3A1, COL5A2, CXCL12, TIMP4, TNC) showed differences in more than three studies, these studies at the transcriptome level are in accordance with the histopathological series that associated endothelial dysfunction, loss of mural cells, inflammatory cell infiltration and degradation of the matrix with sIA wall rupture.MicroRNAs (miRNAs) are a class of small non-coding RNAs, miRNAs are incorporated into the RNA induced silencing complex (RISC) and preferentially bind to the3’untranslated region(3’UTR) of target mRNA. miRNAs have been predicted to regulate20-30%human genes. Recent studies have demonstrated that miRNAs play roles in tumor and cancer.So far, Researchers have not yet identified signaling pathways and key factors for intracranial aneurysm, also failed to simulate phenotype of related genes in animal models. None report has been published about miRNA of intracranial aneurysms. We engaged in clinical work of intracranial aneurysms, and accumulated a large number of experimental results. We found miRNA play an important role in intracranial aneurysms. Test of4cases with miRNA expression profiles in intracranial aneurysm indicated that18miRNA showed differences in expression, we also compared the gene expression of aneurysms vs. tissue of their STAs of two IA patients using Microarray Analysis platform. In all,11351genes showed differences in expression. miRNA centered molecular network developed by IPA identified altogether its mRNA targets, which regulates Vascular wall apoptosis, proliferation and infiltration of inflammatory cells, migration of endothelial cells and smooth muscle cells and macrophage.The project plans to verify pathogenesis of miRNA and its target genes in intracranial aneurysms, and to determine whether they are biologically relevant targets which may have important impact on IA formation. Our study is to explore the regulation of molecular networks of occurrence and development of I As, and to reveal the underlying mechanisms of aneurysm formation.We assess miRNA as prognosis and progression biological markers of the intracranial aneurysm, and a new target for the diagnosis and therapeutic. The research will provide valuable results for the molecular mechanisms of intracranial aneurysms.Methods Results1、To identify miRNAs with significantly different expression levels between LAs and controls.2、To identify mRNAs with significantly different expression levels between I As and controls.3、To predict expression level of miRNA target genes in IAs, and establish molecular regulation system of pathogenesis of intracranial aneurysms.4、To analysis the role and function for miRNA in Cell proliferation, migration, and apoptosis of initiation, growth and rupture of IAs. Results1、 A microarray (Agilent Human miRNA array AMADID1643) study was performed comparing miRNA expression levels in IA tissue samples (n=4) and controls.32miRNAs had significantly different expression. The expression levels of29miRNAs identified in the microarray study were validated using individual real-time qRT-PCR assays,18miRNAs had significantly different expression in IAs.2、A microarray (Agilent Whole Human Genome Oligo Microarray (4×44K)) study was performed comparing mRNA expression levels in IA tissue samples (n=2) and controls.11351mRNAs had significantly different expression.3、Functional classification of the target genes was carried out with IPA,The combined analysis revealed624relation pairs between the miRNAs and mRNAs.4、Ingenuity Pathway Analysis tool was used to generate the network, There were several pathway known to be regulated by these miRNA target genes,including apoptosis of vascular endothelial cell, proliferation and infiltration of inflammatory immune cells, migration of endothelial cells and smooth muscle cells.5、To analysis network established by IPA in pathogenesis of intracranial aneurysms, inflammatory factors plays an important role in the initiation and growth of intracranial aneurysmsConclusion1、miRNA and its target genes play an important role in the initiation and growth of intracranial aneurysms.2、miRNA and target genes regulate the pathogenesis of intracranial aneurysms through mainly several pathway including apoptosis of vascular endothelial cell, proliferation and infiltration of inflammatory immune cells, migration of endothelial cells and smooth muscle cells.3、Cellular immune response and cytokine signaling are the most important pathway in pathogenesis of intracranial aneurysms.更多还原