应用MALDI-TOF IMS技术对弥漫性轴索损伤的初步研究

发布时间：2018-03-19 16:40

本文选题：法医病理学　切入点：弥漫性轴索损伤　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景与目的:弥漫性轴索损伤(diffuse axonal injury,DAI)是指头部遭受钝性外力作用后所产生以脑白质的轴索弥漫性损伤为主要特征的一种脑损伤。由于DAI以脑白质的轴索弥散性损伤为主要特征,缺乏明确的神经系统损害定位体征以及影像学改变,尸检仅凭宏观及一般组织学检查不易诊断,因而DAI是法医学鉴定中面临的一大难题。在损伤诊断和损伤机制研究中,蛋白质和多肽是最常见也是最有效的生物标志物。然而,目前关于DAI的生物标志物十分匮乏,缺乏系列全面的研究。随着人类基因组计划的逐步完成,科学家们开始对基因组表达调控与蛋白质功能进行研究,进一步提出蛋白组计划。对于发现新型的生物标志物和药物,蛋白质组学已经成为非常重要的手段,在临床、基础医学等领域有着非常广泛的应用,也为人类的健康有着很大贡献。近年来,基质辅助激光解吸/电离-飞行时间质谱成像(matrix assisted laser desorption/ionization-time of flight imaging mass spectrometry,MALDI-TOF IMS)技术已成功用于直接分析生物组织切片,作为一项崭新的技术,能够同时绘制组织切片中成百上千个多肽和蛋白的二维质谱图像。MALDI-TOF IMS技术已广泛用于生理和病理组织中蛋白质的表达以及其它分子标志物的发现。以蛋白质组学的方法通过MALDI-TOF IMS技术来识别和筛选生物标志物,能够从整体分析组织的蛋白表达谱,有望发现一系列相关的蛋白质或多肽代替单一分子标志物,以达到DAI早期诊断的目的。方法:DAI动物模型建立和差异蛋白的筛选:参考相关文献制作自由落体打击装置,建立大鼠弥漫性轴索损伤模型,设立大鼠DAI组和对照组,分别取大鼠大脑皮质、大脑髓质、小脑及脑干组织冰冻切片进行MALDI检测,运用布鲁克公司开发的ClinProTools 2.2软件对采集的图谱进行数据分析,获取DAI组织和正常组织的全部蛋白指纹图谱,经统计学分析,初步筛选出DAI大鼠脑组织中特异性表达的蛋白质。DAI组和对照组差异蛋白的质谱成像:参考文献建立DAI大鼠模型,设立DAI组和对照组,获取大鼠脑组织冰冻切片后进行MALDI检测,采集质谱数据,并进行统计学分析,得到两组的差异蛋白,并对差异蛋白进行质谱成像,得到大鼠大脑、小脑、脑干组织切片中差异蛋白的二维分布图像,观察不同的差异蛋白在大脑、小脑及脑干中的分布。实验大鼠DAI诊断模型的建立:参考文献建立DAI大鼠模型,设立DAI组和对照组,将大鼠脑干组织的冰冻切片进行MALDI检测,采集两组的质谱数据,并将两组的质谱数据导入ClinProTools 2.2软件进行统计学分析,运用Supervised Neural Network(SNN)算法建立统计模型,将结果导入 FlexImaging 3.0软件,直接在组织切片上进行成像,将DAI诊断模型可视化,并将诊断模型进行验证。结果:差异蛋白筛选结果:将DAI组和对照组的大鼠脑组织的质谱数据导入ClinProTools 2.2软件进行统计学分析后,通过筛选得到大脑皮质60个差异表达的蛋白质(P0.05),其中有50个在DAI组中表达上调,10个表达下调;大脑髓质56个差异表达的蛋白质(P0.05),其中有46个在DAI组中表达上调,10个表达下调;小脑65个差异表达的蛋白质(P0.05),其中有33个在DAI组中表达上调,32个表达下调;脑干61个差异表达的蛋白质(P0.05),其中有52个在DAI组中表达上调,9个表达下调。质谱成像结果:利用MALDI-TOF IMS采集DAI组和对照组图谱数据,并进行统计学分析后,得到两组的差异蛋白,挑选5个特异性表达的蛋白质在脑组织切片上进行质谱成像,分别为M4963.30Da、M5634.78Da、M6253.65Da、M6714.37Da和M7532.87Da。与对照组相比,M4963.30Da在DAI组小脑中表达减少,在大脑皮质、髓质及脑干中表达增多;M5634.78Da在DAI组大脑皮质及髓质中表达量减少,在小脑和脑干中表达量增多;M6253.65Da在DAI组大脑皮质、髓质及小脑中表达减少,在脑干中表达增多;M6714.37Da在DAI组大脑皮质、髓质及小脑中表达减少,在脑干中表达增多;M7532.87Da在DAI组大脑髓质中表达减少,在大脑皮质、小脑及脑干中表达增多。建立诊断模型结果:将DAI组和对照组的大鼠脑组织的MALDI检测结果导入ClinProTools 2.2软件,并利用SNN算法运算和FlexImaging 3.0软件成像后,得到DAI大鼠诊断模型。诊断模型分为红色和绿色两种颜色,分别代表DAI组和对照组。SNN算法选取了 13个具有特异性表达的蛋白质分子进行建模,特异性较高的蛋白质分子有4个,M7059.21Da和M1518.33Da在DAI组中高表达,而M5077.03Da和M4327.20Da的在对照组中高表达。诊断模型经过验证后,得到整体的交叉验证能力为95.67%,敏感性为99.34%,特异性为92.01%。结论:MALDI-TOF IMS可用于DAI组和对照组大鼠脑组织差异蛋白的筛选,得到上调或下调的差异蛋白质。通过不同质荷比的蛋白质分子进行质谱成像,可立体动态地观察差异蛋白质分子在DAI组和对照组大鼠脑组织中的分布特征。基于MALDI-TOF IMS建立大鼠DAI诊断模型,从而能实现对弥漫性轴索损伤进行诊断的目的。
[Abstract]:Background and objective: diffuse axonal injury (diffuse axonal, injury, DAI) is generated in the cerebral white matter diffuse axonal injury of a brain injury is the main characteristic of the head suffered blunt force. After DAI due to cerebral white matter diffuse axonal injury are the main characteristics. The lack of clear signs of nervous system damage and imaging changes, autopsy alone macroscopic and histological diagnosis is not easy, so DAI is a major problem in forensic medicine. In damage diagnosis and mechanism research, protein and peptide is the most common biomarkers is the most effective yet. At present, about DAI biomarkers are scarce, lack of comprehensive studies series. With the gradual completion of the human genome project, scientists began to study protein function and expression regulation of the genome, further the protein group The plan for the discovery of biomarkers and new drugs, proteomics has become a very important means in clinical, basic medical and other fields have a very wide range of applications, but also for human health has a great contribution. In recent years, matrix assisted laser desorption / ionization time-of-flight mass spectrometry (matrix assisted laser imaging desorption/ionization-time of flight imaging mass spectrometry MALDI-TOF IMS) technology has been successfully used for direct analysis of biological tissue sections, as a new technique,.MALDI-TOF IMS technique of 2D image can draw hundreds of MS in tissue sections of thousands of peptides and proteins have been widely used in protein expression in physiological and pathological tissues and found other molecular markers. With the methods of proteomics by MALDI-TOF IMS technology to identify and screening biomarkers can be divided from the whole Expression analysis of tissue protein, is expected to find a series of related protein or polypeptide instead of single molecular markers, in order to achieve the purpose of early diagnosis of DAI. Methods: DAI animal model and screening of differential proteins: refer to the relevant literature to make a free falling device, establish the rat model of diffuse axonal injury, the establishment of rats of DAI group and control group, were taken from the rat cerebral cortex, cerebral cortex, cerebellum and brainstem tissues were detected by MALDI, using the Brook ClinProTools company to collect map data analysis software for DAI 2.2, and normal tissue of all the protein fingerprint, statistical analysis, preliminary screening of protein group.DAI specific expression in brain tissue of rats in DAI group and the difference of protein mass spectrometry imaging control reference: establish a rat model of DAI established the DAI group and the control group, the acquisition of large Rat brain tissue sections after MALDI detection, mass spectrometry data acquisition, and statistical analysis, get the difference between the two groups of proteins, and the differential proteins by mass spectrometry imaging, rat brain, cerebellum, brainstem slices of two-dimensional distribution of image differences in protein, observe different proteins in the brain, cerebellum and distribution in the brain stem. The establishment of experimental rat model of DAI diagnosis: references to establish the DAI rat models, the establishment of DAI group and control group, the frozen sections of rat brain stem detected by MALDI mass spectrometry data acquisition of two groups, and two groups of mass spectrometry data into ClinProTools 2.2 software for statistical analysis, using Supervised Neural Network (SNN) statistical model algorithm, the results were imported into FlexImaging 3 software, imaging directly in the tissue section, the DAI diagnostic model visualization, and the diagnosis model is verified Results: the difference of protein screening. Results: mass spectrometry data into ClinProTools DAI group and control group rat brain 2.2 software was used for statistical analysis, obtained through screening the cerebral cortex 60 differentially expressed proteins (P0.05), including 50 in group DAI expression, 10 downregulated expression; cerebral medulla 56 differential protein (P0.05), of which 46 were upregulated in DAI group, 10 downregulated expression of the cerebellum; 65 differential protein (P0.05), of which 33 were upregulated in DAI group, 32 downregulated; brain stem 61 differentially expressed proteins (P0.05), of which 52 were upregulated in DAI group, 9 downregulated. Results: using MALDI-TOF mass spectrometry imaging IMS acquisition of DAI group and control group spectrum data, and statistical analysis, get the difference between the two groups of proteins, selected 5 specific protein expression in brain tissue Section of imaging mass spectrometry, respectively M4963.30Da, M5634.78Da, M6253.65Da, M6714.37Da and M7532.87Da. compared with the control group, the expression of M4963.30Da decreased in the DAI group in the cerebellum, cerebral cortex, medulla and brainstem increased expression; the expression of M5634.78Da decreased in DAI group of cerebral cortex and medulla, increased expression in cerebellum and brainstem M6253.65Da; in group DAI, cerebral cortex, cerebellum and medulla decreased expression, increase the expression of M6714.37Da in the brain; in group DAI, cerebral cortex, cerebellum and medulla decreased expression, increased expression in the brain; the expression of M7532.87Da decreased in DAI group of cerebral medulla, in cerebral cortex, cerebellum and brainstem increased expression. The establishment of diagnosis the results of the model: the results of MALDI DAI group and the control group of rat brain tissue into the ClinProTools 2.2 software, and using SNN algorithm and FlexImaging 3 software after imaging, DAI The diagnosis model of rats. The diagnosis model is divided into two colors: red and green, representing the DAI group and control group. The.SNN algorithm selects 13 specific protein molecular modeling, protein molecules with high specificity are 4, M7059.21Da and the high expression of M1518.33Da in group DAI, M5077.03Da and M4327.20Da in the control group. The diagnosis model of high expression after verification, obtained the overall capacity of cross validation was 95.67%, the sensitivity was 99.34%, specificity was 92.01%. conclusion: MALDI-TOF IMS can be used in the DAI group and the control group of rat brain proteins were screened by differential proteins up-regulated or down regulated proteins by mass spectrometry imaging. Different molecular ratio, can dynamically observe the distribution characteristics of three-dimensional protein molecules in the DAI group and the control group in the rat brain. MALDI-TOF IMS rats DAI diagnosis model based on It can be used to diagnose diffuse axonal injury.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：D919.4

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