本文選題：法醫(yī)病理學(xué)　切入點：彌漫性軸索損傷　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景與目的:彌漫性軸索損傷(diffuse axonal injury,DAI)是指頭部遭受鈍性外力作用后所產(chǎn)生以腦白質(zhì)的軸索彌漫性損傷為主要特征的一種腦損傷。由于DAI以腦白質(zhì)的軸索彌散性損傷為主要特征,缺乏明確的神經(jīng)系統(tǒng)損害定位體征以及影像學(xué)改變,尸檢僅憑宏觀及一般組織學(xué)檢查不易診斷,因而DAI是法醫(yī)學(xué)鑒定中面臨的一大難題。在損傷診斷和損傷機制研究中,蛋白質(zhì)和多肽是最常見也是最有效的生物標志物。然而,目前關(guān)于DAI的生物標志物十分匱乏,缺乏系列全面的研究。隨著人類基因組計劃的逐步完成,科學(xué)家們開始對基因組表達調(diào)控與蛋白質(zhì)功能進行研究,進一步提出蛋白組計劃。對于發(fā)現(xiàn)新型的生物標志物和藥物,蛋白質(zhì)組學(xué)已經(jīng)成為非常重要的手段,在臨床、基礎(chǔ)醫(yī)學(xué)等領(lǐng)域有著非常廣泛的應(yīng)用,也為人類的健康有著很大貢獻。近年來,基質(zhì)輔助激光解吸/電離-飛行時間質(zhì)譜成像(matrix assisted laser desorption/ionization-time of flight imaging mass spectrometry,MALDI-TOF IMS)技術(shù)已成功用于直接分析生物組織切片,作為一項嶄新的技術(shù),能夠同時繪制組織切片中成百上千個多肽和蛋白的二維質(zhì)譜圖像。MALDI-TOF IMS技術(shù)已廣泛用于生理和病理組織中蛋白質(zhì)的表達以及其它分子標志物的發(fā)現(xiàn)。以蛋白質(zhì)組學(xué)的方法通過MALDI-TOF IMS技術(shù)來識別和篩選生物標志物,能夠從整體分析組織的蛋白表達譜,有望發(fā)現(xiàn)一系列相關(guān)的蛋白質(zhì)或多肽代替單一分子標志物,以達到DAI早期診斷的目的。方法:DAI動物模型建立和差異蛋白的篩選:參考相關(guān)文獻制作自由落體打擊裝置,建立大鼠彌漫性軸索損傷模型,設(shè)立大鼠DAI組和對照組,分別取大鼠大腦皮質(zhì)、大腦髓質(zhì)、小腦及腦干組織冰凍切片進行MALDI檢測,運用布魯克公司開發(fā)的ClinProTools 2.2軟件對采集的圖譜進行數(shù)據(jù)分析,獲取DAI組織和正常組織的全部蛋白指紋圖譜,經(jīng)統(tǒng)計學(xué)分析,初步篩選出DAI大鼠腦組織中特異性表達的蛋白質(zhì)。DAI組和對照組差異蛋白的質(zhì)譜成像:參考文獻建立DAI大鼠模型,設(shè)立DAI組和對照組,獲取大鼠腦組織冰凍切片后進行MALDI檢測,采集質(zhì)譜數(shù)據(jù),并進行統(tǒng)計學(xué)分析,得到兩組的差異蛋白,并對差異蛋白進行質(zhì)譜成像,得到大鼠大腦、小腦、腦干組織切片中差異蛋白的二維分布圖像,觀察不同的差異蛋白在大腦、小腦及腦干中的分布。實驗大鼠DAI診斷模型的建立:參考文獻建立DAI大鼠模型,設(shè)立DAI組和對照組,將大鼠腦干組織的冰凍切片進行MALDI檢測,采集兩組的質(zhì)譜數(shù)據(jù),并將兩組的質(zhì)譜數(shù)據(jù)導(dǎo)入ClinProTools 2.2軟件進行統(tǒng)計學(xué)分析,運用Supervised Neural Network(SNN)算法建立統(tǒng)計模型,將結(jié)果導(dǎo)入 FlexImaging 3.0軟件,直接在組織切片上進行成像,將DAI診斷模型可視化,并將診斷模型進行驗證。結(jié)果:差異蛋白篩選結(jié)果:將DAI組和對照組的大鼠腦組織的質(zhì)譜數(shù)據(jù)導(dǎo)入ClinProTools 2.2軟件進行統(tǒng)計學(xué)分析后,通過篩選得到大腦皮質(zhì)60個差異表達的蛋白質(zhì)(P0.05),其中有50個在DAI組中表達上調(diào),10個表達下調(diào);大腦髓質(zhì)56個差異表達的蛋白質(zhì)(P0.05),其中有46個在DAI組中表達上調(diào),10個表達下調(diào);小腦65個差異表達的蛋白質(zhì)(P0.05),其中有33個在DAI組中表達上調(diào),32個表達下調(diào);腦干61個差異表達的蛋白質(zhì)(P0.05),其中有52個在DAI組中表達上調(diào),9個表達下調(diào)。質(zhì)譜成像結(jié)果:利用MALDI-TOF IMS采集DAI組和對照組圖譜數(shù)據(jù),并進行統(tǒng)計學(xué)分析后,得到兩組的差異蛋白,挑選5個特異性表達的蛋白質(zhì)在腦組織切片上進行質(zhì)譜成像,分別為M4963.30Da、M5634.78Da、M6253.65Da、M6714.37Da和M7532.87Da。與對照組相比,M4963.30Da在DAI組小腦中表達減少,在大腦皮質(zhì)、髓質(zhì)及腦干中表達增多;M5634.78Da在DAI組大腦皮質(zhì)及髓質(zhì)中表達量減少,在小腦和腦干中表達量增多;M6253.65Da在DAI組大腦皮質(zhì)、髓質(zhì)及小腦中表達減少,在腦干中表達增多;M6714.37Da在DAI組大腦皮質(zhì)、髓質(zhì)及小腦中表達減少,在腦干中表達增多;M7532.87Da在DAI組大腦髓質(zhì)中表達減少,在大腦皮質(zhì)、小腦及腦干中表達增多。建立診斷模型結(jié)果:將DAI組和對照組的大鼠腦組織的MALDI檢測結(jié)果導(dǎo)入ClinProTools 2.2軟件,并利用SNN算法運算和FlexImaging 3.0軟件成像后,得到DAI大鼠診斷模型。診斷模型分為紅色和綠色兩種顏色,分別代表DAI組和對照組。SNN算法選取了 13個具有特異性表達的蛋白質(zhì)分子進行建模,特異性較高的蛋白質(zhì)分子有4個,M7059.21Da和M1518.33Da在DAI組中高表達,而M5077.03Da和M4327.20Da的在對照組中高表達。診斷模型經(jīng)過驗證后,得到整體的交叉驗證能力為95.67%,敏感性為99.34%,特異性為92.01%。結(jié)論:MALDI-TOF IMS可用于DAI組和對照組大鼠腦組織差異蛋白的篩選,得到上調(diào)或下調(diào)的差異蛋白質(zhì)。通過不同質(zhì)荷比的蛋白質(zhì)分子進行質(zhì)譜成像,可立體動態(tài)地觀察差異蛋白質(zhì)分子在DAI組和對照組大鼠腦組織中的分布特征�；贛ALDI-TOF IMS建立大鼠DAI診斷模型,從而能實現(xiàn)對彌漫性軸索損傷進行診斷的目的。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：D919.4

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