【作者】 张建鹏；

【导师】 焦炳华；

【作者基本信息】 第二军医大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 神经组织退行性疾病在现今快速老龄化的发展中国家及发达国家常见疾病谱里占有相当大的比重,如阿尔茨海默病（AD）、帕金森病（PD）和亨廷顿舞蹈病（HD）等。据世界帕金森病研究协会的资料显示,PD已经成为继AD（老年性痴呆）之后第二号神经疾病杀手。大多数PD患者于50～60岁发病,国外男性发病率高于女性（2:1）,而我国的资料也表明,男性患者明显多于女性（2～3:1）。中国目前有超过200万的PD病患,这一数字远远超过了美国（少于100万人）。尽管中国人口统计学的变化及老龄化趋势不同于西欧和北美,但是中国正面临着大量的PD病例,预计在未来的十年里PD带来的负担将不断加大。PD是一种慢性中枢神经系统失调,是中老年人中常见的神经退行性疾病之一。其症状发展一般比较缓慢,发展的顺序各患者之间不尽相同,其主要特征为黑质纹状体通路的多巴胺能神经元的选择性变性。PD典型的病理学改变是包括α-synuclein和泛肽在内的胞浆内含物路易氏体（Lewy body）的出现。近几年,随着蛋白质组学及其相关技术的快速发展,质谱技术已广泛应用于PD发病机理的研究中,并找到了一些PD相关的特异性蛋白质,如α-synuclein和DJ-1等。但是,到目前为止PD的发病机理还不是十分明确。因此,寻找PD检测与监控的生物标志物对揭示PD发病机理具有重要意义。蛋白质糖基化是其翻译后重要的修饰方式之一,对细胞的正常生长起了重要的调节作用,直接或间接地影响着蛋白质的生物学功能。然而,蛋白质的异常糖基化会导致人类疾病的发生发展,如PD等。糖蛋白质组,作为蛋白质组的一个重要分支,涵盖了所有糖蛋白/糖肽的图谱与信息,并携带了许多潜在的疾病诊断和发病过程监控的生物标志物。目前在PD研究中,糖蛋白的发展还处于起步阶段,现代质谱技术的应用对糖蛋白的分析鉴定以及潜在生物标志物的筛选起到了很大的推动作用。本课题采用酰肼树脂法分离富集了正常人、不同程度PD患者和AD患者脑组织和脑脊液中的糖蛋白,用iTRAQ试剂对胰酶酶解后的糖肽进行定量标记,利用反相色谱和MALDI-TOF/TOF质谱技术鉴定并获得了人脑组织和脑脊液中糖蛋白谱,并对PD及其发展过程中潜在的糖蛋白标志物进行了初步筛选。研究内容:1.帕金森病及其发展过程人脑组织中糖蛋白谱的定性与定量分析;2.帕金森病及其发展过程人脑脊液中糖蛋白谱的鉴定;3.帕金森病及其发展过程中候选糖蛋白标志物的初步筛选。研究方法:1.样品取样与分组:（1）根据PD及其发展过程的病理诊断结果,设定了四个脑组织样品组（每组5例样本）,取样区域为中前部脑回区,匀浆后进行蛋白定量测定。①CTL组:无病史、症状或精神病和神经病的正常对照脑组织样品;②PD-B组:病理检测为仅脑干出现LB的脑组织样品（Braak分级≤1）;③PD-L组:脑干及脑边缘出现LB的脑组织样品（Braak分级1-2级）;④PD-I组:大脑新皮层、脑干及脑边缘出现LB的脑组织样品（Braak分级4-5级）（2）从腰椎穿刺获得CSF样品,经无血污染检测后对样品进行分组。①CTL组:无病史、症状或精神病和神经病的正常对照CSF样品（29例）;②AD组:经综合临床检验确诊为AD（美国NINCDS ADRDA标准）（51例）;③PDE组:早期PD组,经综合临床检验确诊为PD（Hoehn-Yahr分级≤1.5级,11例）;④PDL组:晚期PD组,经综合临床检验确诊为PD（Hoehn-Yahr分级≥3级,11例）2.人脑组织与脑脊液样品中总糖蛋白的分离纯化:（1）各组取等量蛋白用TCA/丙酮法沉淀总蛋白质;（2） BCA法测定脑组织匀浆液与脑脊液样品总蛋白含量;（3）脑组织与脑脊液总蛋白的胰酶酶解;（4） SDS-PAGE结合银染检验酶解完全程度;（5）脑组织与脑脊液总蛋白酶解片段的iTRAQ标记;（6） iTRAQ标记肽段的除盐、氧化与纯化;（7）酰肼树脂法富集经iTRAQ标记的糖肽:标记肽段与预处理酰肼凝胶孵育后经PNGase F酶切,获得纯化的N-糖肽。3.人脑组织与脑脊液糖肽的质谱分析:（1）待测样品用LC Packing Ultimate^TM液相色谱系统进行反相色谱分离后点板;（2） 4800 plus MALDI-TOF/TOF^TM质谱仪自动采集数据;（3）采用ProteinPilot^TM软件和EBI的IPI-人蛋白数据库对所得MS/MS图谱进行分析;（4）鉴定的糖蛋白与UniProtKB/Swiss-Prot数据库和ISB数据库中已知或可能/潜在的糖基化位点的糖蛋白进行校对;（5）应用GO分析对所得糖蛋白进行分类分析。4.应用western blotting法对筛选出的表达差异的候选糖蛋白标志物在血浆中做进一步的验证:（1）血浆样品分为正常对照、早期PD、中期PD以及晚期PD四组;（2）酰肼树脂法富集血浆总糖蛋白并进行蛋白浓度测定;（3）候选糖蛋白标志物在血浆样品中的western blotting分析。结果:1.根据BCA蛋白浓度测定结果,每组脑组织与脑脊液样品取120μg进行定性定量分析;2.银染结果显示脑组织与脑脊液样品总蛋白胰酶酶解完全;3.反相色谱图谱显示脑组织与脑脊液中iTRAQ标记糖肽分离效果良好,有助于质谱分析鉴定;4.采用ProteinPilot^TM软件和EBI的IPI-人蛋白数据库,对所得MS/MS图谱进行分析,分别找到了人脑组织和脑脊液中394种和283种非冗余糖蛋白。5.将所得糖蛋白数据与UniProtKB/Swiss-Prot和ISB的糖蛋白数据库进行比较后发现,人脑组织中有343种糖蛋白能够在这两个数据库中找到,特异性为87%（343/394）,而人脑脊液中有243种糖蛋白能够在这两数据库中找到,特异性为86%（243/283）。所有鉴定的糖蛋白/糖肽经ProteinPilot^TM软件所得报告数据的可信度达到了95%（ProtScore≥1.3）。6.GO分析对人脑组织和脑脊液中糖蛋白进行细胞组分和生物过程分类,发现多数糖蛋白为细胞外蛋白和膜蛋白,其中人脑组织中糖蛋白以膜蛋白为主（IPIs=0.612）,而人脑脊液中糖蛋白以细胞外蛋白为主（IPIs=0.606）。7.对人脑组织和脑脊液中糖蛋白谱进行对比分析,发现98种重叠蛋白（各占25%和40%）,其中与CNS结构及功能相关的重叠蛋白有43种。8.对正常人和不同程度PD患者人脑组织中糖蛋白进行定量分析后发现,有43种糖蛋白的含量在这些样本中发生了明显的变化。9.Western blotting结果显示,无论是在分组试验还是在个体试验中,有6种候选糖蛋白的表达变化十分显著。其中4种糖蛋白表达上调,如CP、TNC、VCAN和MAG,2种表达下调,如NPTXR和GRIN2B。结论:本课题首次对正常人和PD患者脑组织以及脑脊液中糖蛋白谱进行了系统性的定性与定量分析,分别鉴定了394种和283种糖蛋白。研究发现,两种样品中有43种与中枢神经系统结构及功能相关的重叠蛋白。脑组织中鉴定得到的糖蛋白有43种存在表达差异,其中6种候选糖蛋白在血浆中表达变化显著,提示其很可能是PD及其发展过程中潜在的生物标志物。本课题为PD发病机理的阐明及疾病的检测和监控提供了重要的理论和实验资料。更多还原

【Abstract】 PD is traditionally considered a movement disorder that results from a relatively selective loss of neurons in the brainstem,including dopaminergic neurons in the substantia nigra pars compacta,with subsequent loss of striatal dopamine and accompanied by the formation of intraneuronal inclusions called Lewy bodies that containα-synuclein as one of the major proteins.PD is the forth common disease occured in senior citizens.Although some unique proteins,such asα-synuclein and DJ-1, were studied,the pathogenesis of PD is still unknown.Additionally,selection of biomarkers that can predict and examine PD and its progression is important as well.Protein glycosylation regulates protein function and cellular distribution.Furthermore, aberrant protein glycosylations have been recognized to play major roles in human disorders,including PD and AD.Glycoproteomics,a branch of proteomics that catalogs and quantifies glycoproteins,provides a powerful means to systematically profile the glycopeptides or glycoproteins of a complex mixture,and therefore,cany great potential to be diagnostic and/or prognostic biomarkers.Until recently,very little has been known about the role of glycosylated proteins in PD.Application of this mass spectrometry-based technology will improve the identification and selection of glycoprotein biomarkers in PD and its progression.In this study,hydrazide resin was used to enrich glycoproteins which were isolated from brain tissue and CSF of normal people and PD patients.To perform quantitative analysis of glycoproteins unique to PD and PD progression,samples were digested with trypsin,followed with iTRAQ labeling.With MALDI-TOF/TOF,the glycoproteins/glycopeptides spectra were extracted and searched against the IPI human protein database with ProteinPilot^TM software with the Paragon^TM method.The raw peptide identification results flom the Paragon^TM Algorithm（Applied Biosystems） searches were further processed with the Pro Group^TM Algorithm within the ProteinPilot^TM software before final display.The Pro Group Algorithm uses the peptide identification results to determine the minimal set of proteins that can be reported for a given protein confidence threshold.The MS/MS analysis revealed a total of 394 non-redundant glycoproteins in human brain tissue and 283 non-redundant glycoproteins in hurnan CSF.In comparison with the existing publicly accessible database,343 of human tissue glycoproteins and 243 of human CSF glycoproteins were annotated in UniProtKB/Swiss-Prot and the ISB database as glycoproteins with known glycosylation sites or probable/potential glycosylation sites.The specificity of this approach were 87%（343/394） and 86% （243/283） separately.All reported data were based on 95%confidence for protein identification as determined by ProteinPilot（ProtScore≥1.3）.Additionally.when the glycoproteins identified in human brain tissues and CSF were classified by GO analysis,it was apparent that a majority of the proteins belong to either the extracellular compartment or are associated with the plasma membrane.This is entirely consistent with the claim that most membrane proteins are glycosylated,and that a significant portion of glycoproteins are designated for secretion into the extracellular fluid and thereby enter blood or CSF.In this study,98 proteins were seen in brain tissue（a total of 394 proteins） and CSF（a total of 283 proteins） to account for 25% of brain tissue glycoproteins and 40%of CSF glycoproteins,besides 43 proteins are associated with CNS.These overlapping proteins identified with glycoprotein isolation are more likely related to PD pathogenesis.With quantitative analysis,more than 40 glycoproteins were changed in normal brain tissue and PD patients’ brain tissue,which indicates that these glycoproteins might be the potential biomarkers in PD and its progress.For further study,the total plasma glycoproteins were isolated from normal people,PD in different stage and AD patiens by hydrazide resin to perform western blotting analysis.The result shows that 6 unique glycoproteins have notable expression both in group samples and in sigle samples.Thus, these candidate glycoproteins might be the potential biomarker in PD and its progression.It should be emphasized that this dataset represents the first systematic analysis of glycoproteins in human brain in normal and diseased settings.This study identificated and selected several glycoproteins as potential biomarker in PD and its progress,which provided important imformation and evidence to illustrate PD pathogenesis.更多还原