【作者】 王芳芳；

【导师】 姬新颖；

【作者基本信息】 河南大学 ， 免疫学， 2014， 硕士

【摘要】 背景动脉粥样硬化(atherosclerosis AS)是动脉硬化中最常见、最重要的一种，因其基本损害从动脉内膜开始局部呈斑块状增厚，也被称为动脉粥样硬化性斑块。动脉粥样硬化发病率很高，近年来呈增长趋势。它是一种复杂的多因素疾病，其发病机制和确切病因尚未完全阐明。动脉内皮细胞层能感知血液中的切应力、压力、炎性信号、激素水平等信息，通过释放活性物质对这些信息作出反应，维持血管壁张力、血液流动、屏障、管壁的炎症修复和血管增生等功能，其作为动脉粥样硬化开始的起始部，内皮细胞的功能障碍或损伤在动脉粥样硬化中起到了重要的作用。冠状动脉近侧段好发动脉粥样硬化可能与内皮异质性相关，长期环境因素的影响可能使不同部位的内皮细胞基因表达不同。目前，关于不同种类动脉粥样硬化与血管内皮基因表达的研究主要偏重于特定的基因，从整体研究基因表达的改变并不多见。基因芯片可以用少量样品在一次试验中同时平行分析成千上万个基因，从整体宏观的角度研究生物体基因的表达及功能。目前,基因芯片技术已广泛应用于基因测序、基因表达谱分析、基因突变及多态性分析、新基因的发现、药物筛选、疾病诊断和分型等。本课题主要利用基因芯片技术分析冠状动脉内皮细胞与主动脉内皮细胞基因表达谱，筛选出表达差异的基因，探讨相关的功能和信号通路。目的芯片技术分析冠状动脉内皮细胞与主动脉内皮细胞基因表达谱，筛选出表达差异的基因，探讨相关的功能和信号通路。方法提取冠状动脉内皮细胞（HCAEC）与主动脉内皮细胞（HAEC）株的总RNA，纯化后逆转录制备杂交探针，采用Agilent公司人基因组寡聚核苷酸芯片筛选差异表达基因，然后采用DAVID功能注释生物信息学芯片分析系统对P值小于0.05且差异表达大于2倍的基因进行基因富集分析，并用实时定量PCR、免疫印迹(Western blot)验证部分筛选基因的表达。选取基因EDN1、OCLN构建过表达质粒，将重组质粒通过脂质体转染到HCAEC中，用G418筛选出稳定株，用Western blot方法验证并检测过表达后对部分基因的影响。然后用四甲基偶氮唑盐比色法（MTT）作出生长曲线图，用流式细胞术检测细胞周期，探讨EDN1、OCLN对细胞增殖的影响。结果与主动脉内皮细胞相比，冠状动脉内皮细胞差异表达在两倍以上，P值小于0.05的差异点有2435个，其中1198个基因上调，1237个基因下调。GO分类后可见差异表达基因涉及细胞增殖、分化、粘附、炎症反应、脂代谢、免疫反应、宿主防御、应激反应、离子转运以及血管生成和细胞凋亡等多个基因及信号通路；Pathways分析后可以得出参与较多基因的信号通路，主要涉及肿瘤途径、细胞因子间受体相互作用、MAPK信号途径、粘附分子、细胞骨架调节、内吞作用、ECM受体相互作用、Jak-STAT信号途径等。实时定量PCR验证的部分基因与芯片结果一致。成功构建了EDN1、OCLN过表达质粒并稳定转染到HCAEC中，在蛋白水平，Western blot结果显示过表达后的表达量增高。MTT结果显示过表达后对HCAEC的生长抑制，用流式细胞术检测细胞周期显示S期细胞减少，通过细胞周期抑制增殖。结论应用表达谱基因芯片成功筛选出冠状动脉内皮细胞与主动脉内皮细胞差异表达基因，经生物学信息分析主要涉及细胞增殖、分化、粘附、炎症反应、脂代谢、免疫反应、宿主防御、应激反应、离子转运以及血管生成和细胞凋亡等多个基因及信号通路，可能为研究冠状动脉粥样硬化发病机制及防治提供新思路。基因EDN1、OCLN过表达后对冠状动脉内皮细胞增殖的影响可能与细胞周期阻滞相关。更多还原

【Abstract】 IntroductionAtherosclerosis (AS) is the most common and the most important kind of atherosclerosis, because itsbasic damage starts from patchy thickening of the arterial intima locally, it’s also called atheroscleroticplaques. Atherosclerosis exists with high incidence,with a growing trend in recent years. It is a complexmultifactorial disease, its pathogenesis and the exact cause is not fully understood. Arterial endothelial celllayer can apperceive stress sensing blood pressure, inflammatory signals, hormones and other informationthrough the release of the active substance, react to this information, maintain vascular wall tension,bloodflow,barrier wall repair and inflammation angiogenesis and other functions.As the beginning part ofatherosclerosis,endothelial dysfunction or injury play an important role in atherosclerosis. The proximalsegment of coronary atherosclerosis may be associated with endothelial heterogeneity.Environmentalfactors may affect long-term endothelial cell expression of different genes in different parts.Currently, studyof different types of atherosclerosis and vascular endothelial basement mainly emphasis on the expressionof specific genes, studies of changing the overall gene expression are rare.Gene chips can be analyzed inparallel with a small sample of thousands of genes simultaneously in a single experiment,studying theexpression and function from the angle of the overall macroscopic objects graduate gene. Currently, genechip technology has been widely used in gene sequencing, gene expression profiling, gene mutation andpolymorphism analysis, discovery of new genes, drug screening, diagnosis and typing and so on. Thispaper uses gene chip to analysis gene expression profiling of coronary artery endothelial cells and aorticendothelial cell, screens out gene expressing differences, and explores the related functions and signalingpathways.ObjectiveUsing gene chip to analysis gene expression profiling of coronary artery endothelial cells and aorticendothelial cell, screens out gene expressing differences, and explores the related functions and signalingpathways MethodTotal RNA was extracted from coronary artery endothelial cells (HCAEC) and aortic endothelial cells(HAEC) strains, hybridization probe was prepared and purified by reverse transcription, using Agilent’shuman genome oligonucleotide microarray screening out differentially expressed genes, and then using theDAVID functional Notes bioinformatics microarray analysis system for P <0.05and greater than2-folddifference in gene expression for gene enrichment analysis and real-time quantitative PCR, immunoblotting(Western blot) partially screened gene expression validation. Building recombinant plasmid of EDN1,OCLN overexpression and stably transfected into HCAEC by liposome and screened with G418, verifiedby Western blot method to detect the implications for the rear portion of the gene expression. Then makeuse of MTT assay growth curve,test cell cycle by flow cytometry, explore impact of EDN1, OCLN on cellproliferation.ResultCompared with aortic endothelial cells, coronary artery endothelial cells express more than twice thedifference, P value less than0.05points of difference have2435, of which1,198genes were up regulated,1237genes were down regulated. By GO classification Visible differences in expression of genes involvedin cell proliferation, differentiation, adhesion, multiple genes and signaling pathways involved ininflammation, lipid metabolism, immune response, host defense, stress response, ion transport as well asangiogenesis and apoptosis; Pathways analysis of the gene can be obtained more signaling pathwaysinvolved, mainly related cancer pathways between cytokine receptor interaction, MAPK signalingpathways, adhesion molecules, cytoskeletal regulation, endocytosis, ECM receptor interaction, Jak-STATsignaling pathway and so on. Validation part gene consistent with the results of gene chip by real-timequantitative PCR. Successfully constructed EDN1, OCLN overexpression vector and stably transfected intoHCAEC, and at the protein level, Western blot showed that the gene expression level increased. MTTresults showed that HCAEC growth inhibition after over-expression of gene, flow cytometry display S-phase cells decreased,it may inhibit the cell proliferation by cell cycle.ConclusionsApplication of Gene Expression Profiling successfully screens out the differences coronary and aorticendothelial cell gene expression in endothelial cells.By biological information analysis,it mainly involvescell proliferation, differentiation, adhesion, inflammation, lipid metabolism, immune response, host defense, stress multiple genes and signaling pathways reactions, ion transport as well as angiogenesis and apoptosis,it may bring new ideas for study of the pathogenesis of coronary atherosclerosis and its prevention.Thereare affect on proliferation of coronary artery endothelial cell after gene EDN1, OCLN overexpression,itmay be associated with cell cycle arrest.更多还原