阿尔茨海默病病因的基因表达谱数据分析

发布时间：2017-12-27 06:34

本文关键词：阿尔茨海默病病因的基因表达谱数据分析　出处：《南方医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：阿尔茨海默病是一种常见的慢性、进行性中枢神经系统退行性疾病,其临床主要表现为进行性的记忆衰退和认知功能的下降、行为异常和社交障碍,通常病情呈进行性加重,病程缓慢不可逆。随着社会人口的老龄化,阿尔茨海默病的发病率呈上升趋势。据统计数据显示,老年人阿尔茨海默病(Alzheimer's Disease, AD)的患病率仅次于心血管病和癌症居第三位,全世界有近3600万人正在遭受着AD的折磨。目前阿尔茨海默病还是绝症,没有任何治疗方法可以使患者痊愈。阿尔茨海默病给社会和家庭带来沉重的负担,给患者本人也带来精神上和身体上的极大痛苦。阿尔茨海默病可发生在成年人的任何年龄,发病率随年龄的增长而成指数型增长。女性的发病率较男性高,农村人发病率较城市人高,受教育程度高的人群发病率相对较低。AD的致病危险因素还包括受教育程度低、头部外伤、性激素水平降低、家族遗传史、吸烟、抑郁症状、心理压力、血管因素、体温较低、社会活动较少、轻度认知损伤以及病毒感染等。阿尔茨海默病病灶一般先出现在大脑边缘系统的海马区、内嗅皮层等部位,随后蔓延到颞叶、扣带回、顶叶、额叶等大脑皮层区,一般波及不到枕叶、小脑、运动和感觉皮层区域。阿尔茨海默病人大脑的病理病变有：神经炎性老年斑(senile plaques,SPs),神经原纤维缠结(neurofibrillary tangles,NFTs),神经细胞和突触的消失,脑淀粉样血管病以及颗粒空泡变形等,整体表现为弥漫性大脑皮质的萎缩和脑室的扩大。阿尔茨海默病的发病机制仍然不清楚。目前有分子遗传和基因学说、炎症学说、自由基学说、胆碱能学说、金属离子假说、病毒学说、氧化应激学说、雌激素水平降低学说等。大多数研究犹如盲人摸象,分不清哪些机制是病因哪些是造成的结果。因此很多研究所和大学机构也投入了相当多的人力物力来研究AD的发病机制,但效果很不理想。目前需要从整体宏观上来认识这个疾病,而且不能受到各个机制的单独片面影响。需要从基因表达上入手,以基因表达差异的客观现象来了解和研究AD的发病机制、治疗手段以及早期检测方法。由于神经细胞的复杂,而且取样有限,从基因表达水平筛选出和年龄相关的基因,成为阿尔茨海默病分子发病机理研究的最佳途径。近年发展起来的基因芯片技术,显示了其在功能相关差异基因筛选方面的优越性,少量取样就能获得海量的基因表达数据,这将增强对阿尔茨海默病了解,而且得到的数据更为客观,具有较高的整体性。目前,由于高通量基因芯片技术的广泛应用,各个研究所和实验室产生出了大量的实验数据,但是这些数据的信息只用到了小部分,全部信息往往没有被完全挖掘出来。美国国立生物技术信息中心(National Center for Biotechnology Information, NCBI)建立的GEO (Gene Expression Omnibus)数据库是当前规模最大、收录最全、免费开放的公共基因表达数据库,包含大量的、各种各样的复杂生物数据。这些数据从表面上看起来是杂乱无章,无可下手,但是海量的生物学数据中必然蕴含着重要的生物学规律,这些规律将是解释阿尔茨海默病病因的关键。基因表达谱数据庞大复杂,变量数以万计。为此,通过使用Qlucore Omics Explorer (QOE) 3.0生物信息学软件,把数据经统计计算,快速转换为3D可视化图,再经过主成分分析(Principle Component Analysis, PCA)和聚类分析,识别出数据中隐藏的结构和模式,以标准的统计学方法得到样本与样本之间、组与组之间以及数据集与数据集之间的差异与联系。筛选出共同或差异的基因,再通过Database for Annotation, Visualization and Integrated Discovery (DAVID)6.7在线基因注释系统,对基因列表进行Gene Ontology (G O)功能富集分析,从Cellular Component (CC),Biological Process (BP),Molecular Function (MF)三个方面对基因涉及的功能进行注释、富集、聚类。得出AD患者生物学过程、细胞组成以及分子功能方面上的差异和变化。因此,本人根据目前现有的人类阿尔茨海默病基因表达谱数据集,从整体的基因表达差异、随年龄增长的基因表达差异、各个脑区的基因表达差异、不同发病阶段的基因表达差异、性别相关的基因表达差异入手,对阿尔茨海默病的发病机制进行探究。首先,使用数据集GSE36980,通过两组比较,筛选出阿尔茨海默病患者和正常人的大脑的差异基因196个。对差异基因进行表达和功能的聚类分析,结果分为42个注释集群,涉及的GO主要有蛋白质定位、离子转运、细胞信号、学习记忆、披网格蛋白小泡、突触、细胞质膜、细胞连接、线粒体、钙离子绑定、离子通道、tau蛋白激酶以及脂肪酸转运等。这些差异功能都和阿尔茨海默病临床表现以及病理有一定的联系。这些结果证明并支持了阿尔茨海默病大脑内神经元的蛋白体破坏补偿机制,神经元突触大量减少而且其功能受到严重影响,同时又发现了高尔基体在阿尔茨海默病患者的大脑中都会形成碎片的原因和相关差异基因。阿尔茨海默病的神经元细胞膜遭到了破坏,线粒体功能的异常使细胞能量供应受损,并产生过量的自由基。阴离子通道和金属阳离子通道异常,致使细胞信息传递和信号通路的错误发生。阿尔茨海默病人的学习记忆相关基因及生物学进程发生变化,造成了记忆和认知功能的下降。细胞骨架及微管也发生异常并导致神经元的立体空间结构和连接遭到破坏,其中磷酸化过程也出现异常,这将使tau蛋白过度磷酸化,并形成神经元纤维缠结。为了筛选出和年龄因素相关的阿尔茨海默病易感基因,本人同时使用GSE36980和GSE53890数据集,通过线性回归和两组比较结合,筛选出来20个在阿尔茨海默病和正常人中有差异又在年龄上有线性趋势的基因。差异基因涉及的GO生物学过程主要有蛋白质代谢、细胞周期、神经代谢正调控、轴突、质膜、突触、细胞骨架、和金属离子绑定。这些功能随着年龄增长而减弱是阿尔茨海默病发病率和年龄有正相关性的原因,这也说明了正常人年龄的增加会导致大脑萎缩,大脑相关功能减弱,并出现类阿尔茨海默的相关症状。不同大脑区域的阿尔茨海默病发病率也不一样。此部分使用GSE36980和GSE9770这两个数据集,通过两组比较和多组比较,筛选出11个在各个脑区差异表达的阿尔茨海默病易感基因。这些基因的功能主要涉及细胞极性的建立或维护、离子绑定、质膜功能、钙离子绑定,这些功能都和信号传导和信息传递功能有关。边缘系统的这些功能较其他区域活性较高,这是边缘系统易受疾病的影响重要原因,也是阿尔茨海默发病后先出现学习、情绪和记忆障碍的原因。其中有两个基因CBLN4和LRRTM1也是年龄相关的阿尔茨海默差异表达基因。通过秩回归分析GSE1297数据集,筛选出了随病程加剧而变化的基因,该数据集是不同病程的海马区神经元的基因表达谱数据。结果中有65个基因被筛选出来,其中32个随疾病加重不断上调表达,33个基因随疾病加重下调表达。其中上调的基因涉及的GO功能包括转录调节、质膜组分等。下调的基因涉及GO功能有线粒体功能、蛋白质定位和运输、核苷酸绑定等。因此,对于不同发病程度的AD患者,应根据其生物学相关功能的改变采取个性化的治疗方法以及精准的药物剂量。男性女性的大脑基因表达有一定的差异,为探究阿尔茨海默病女性发病率男性高的基因表达层面上原因,研究采用了GSE36980数据集,并选用数据集中的阿尔茨海默病组,在组内进行性别的两组比较,在满足统计学意义的前提下筛选出了26个男女大脑表达差异基因。这些基因涉及的GO生物学功能包含细胞骨架、离子传递、蛋白质运输与定位、核苷绑定以及膜功能等。这些功能的相关基因表达女性比男性较低,是女性较男性易感阿尔茨海默的原因之一。其中差异基因列表中还包含CBLN4基因,说明此基因在阿尔茨海默病的发病机制中起着非常重要的作用。以上所有分析是以基因表达水平上对阿尔茨海默病进行研究,从整体水平上的流行病学统计结果和病理现象找出差异基因,并分析差异变化的生物学功能。把宏观和微观联系起来,为阿尔茨海默病的预防、诊断、研究和治疗提供客观的理论支持。
[Abstract]:Alzheimer's disease is a common chronic progressive neurodegenerative disease of the central nervous system. Its main clinical manifestations are progressive memory impairment and cognitive decline, behavioral abnormalities and social barriers. Usually, the disease is progressively aggravated, and the course of disease is slow and irreversible. With the aging of the population, the incidence of Alzheimer's disease is on the rise. According to statistics, the prevalence of Alzheimer's Disease (AD) is third only in the elderly, ranking the top 36 million in cardiovascular disease and cancer. Nearly 36 million people in the world are suffering from AD. At present, Alzheimer's disease is still a disease, and there is no cure for the patient. Alzheimer's disease brings a heavy burden to the society and the family, and brings great mental and physical pain to the patient himself. Alzheimer's disease can occur at any age of adults, and the incidence of Alzheimer's disease increases exponentially with age. The incidence of women is higher than that of men. The incidence of rural people is higher than that of urban people, and the incidence of high educated people is relatively low. The risk factors of AD include low education level, head trauma, sex hormone levels, family history, smoking, depressive symptoms, psychological pressure, vascular factors, lower body temperature, less social activities, mild cognitive impairment and viral infection. Alzheimer's disease usually first appeared in the hippocampus and entorhinal cortex of the limbic system, and then spread to the cerebral cortex of temporal lobe, cingulate gyrus, parietal lobe, frontal lobe and so on, which usually did not reach the occipital lobe, cerebellum, motor cortex and sensory cortex. Pathological changes of the patient's brain: Blzheimer neuritic senile plaques (senile plaques, SPs), neurofibrillary tangles (neurofibrillary, tangles, NFTs), nerve cells and synapses disappear, cerebral amyloid angiopathy and granulovacuolar deformation, the overall performance of diffuse cortical atrophy and ventricular enlargement. The pathogenesis of Alzheimer's disease is still unclear. At present, there are molecular genetic and genetic theory, inflammation theory, free radical theory, cholinergic theory, metal ion hypothesis, virus theory, oxidative stress theory, estrogen level reduction theory and so on. Most of the research is not clear what the mechanism on the basis of one-sided viewpoint, which is the result of cause. Therefore, a lot of research institutes and university institutions have also invested considerable manpower and material resources to study the pathogenesis of AD, but the effect is not ideal. At present, it is necessary to understand the disease from the overall macro level, and can not be affected by the individual and one-sided impact of the various mechanisms. It is necessary to start with the gene expression, to understand and study the pathogenesis, treatment and early detection methods of AD with the objective phenomenon of gene expression difference. Because of the complexity of neurons and limited sampling, screening age-related genes from gene expression level is the best way to study the molecular pathogenesis of Alzheimer's disease. Gene chip technology developed in recent years, shows the superiority in the screening of gene function related differences, a small sample can get massive gene expression data, which will enhance the understanding of Alzheimer's disease, and the data obtained are more objective, highly integrated. At present, due to the extensive application of high-throughput gene chip technology, various research institutes and laboratories have produced a lot of experimental data, but the information of these data is only used to a small part, and all information is often not fully excavated. The National Center for Biotechnology Information (National Center for Biotechnology Information NCBI GEO (Gene Expression) to establish the Omnibus database) is the largest collection of the most complete, free and open public gene expression database, complex biological data contains a lot of variety. These data seem to be disorganized and unavailable on the surface. However, massive biological data are bound to contain important biological laws. These laws will be the key to explain the cause of Alzheimer's disease. The gene expression profiles are huge and complex, with tens of thousands of variables. Therefore, through the use of Qlucore Omics Explorer (QOE) 3 bioinformatics software, the data by statistical analysis, the rapid conversion of 3D visualization, through principal component analysis (Principle Component, Analysis, PCA) and cluster analysis, to identify hidden in the data structure and model, with standard statistical methods and contact the difference between the sample and the sample groups and data sets and data sets. Selected common or different genes, and then through the Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.7 online gene annotation system, Gene Ontology (G O) on the list of gene function enrichment analysis, from Cellular Component (CC), Biological Process (BP), Molecular Function (MF) on genes involved in three the function of annotation, enrichment, clustering. The differences and changes in the biological process, cell composition and molecular function of AD patients were obtained. Therefore, I according to the current human Alzheimer's disease gene expression data sets available, from the overall gene expression difference, with the increase of age differences in gene expression, gene expression differences in various brain regions, different stages of gene expression differences, sex related gene expression differences of pathogenesis of Alzheimer's disease research. First, using the data set GSE36980, 196 different genes of the brain of Alzheimer's disease patients and normal people were screened by two groups. The cluster analysis of expression and function of differentially expressed genes is divided into 42 annotation clusters, including GO protein localization, ion transport, cell signaling, learning and memory, and clathrin.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R749.16;Q78

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