【作者】 姜寿峰；

【导师】 丁美萍；

【作者基本信息】 浙江大学 ， 神经病学， 2011， 博士

【摘要】 研究背景：多发性硬化(Multiple sclerosis, MS)及视神经脊髓炎(Neuromyelitis optica, NMO)均是病因不明的中枢神经系统免疫性脱髓鞘疾病,病理过程主要是脱髓鞘改变,临床表现依中枢神经系统累及的部位不同而呈现多样化。多发性硬化及视神经脊髓炎虽同属中枢神经系统脱髓鞘病,但两者在发病机制、病理表现、临床特点、治疗方案等方面有诸多不同,尤其是在NMO患者血清中发现自身抗体NMO-IgG后,目前更倾向NMO并非是MS的一种亚型而是一个独立的疾病。但由于中枢神经系统组织标本的不易获取,阻碍了对MS及NMO病理机制的进一步研究,而体液作为内环境的重要组成部分,其所含有的多肽、蛋白质和酶类在生理过程中起到非常重要的作用,其蛋白与多肽的质和量的变化,能精确反映机体疾病状态下的病理生理过程,MS及NMO复杂的免疫应答引起脑脊液、血清、脑组织、各种亚细胞器中蛋白质种类和数量的动态变化。鉴于此,可以通过研究MS和NMO血清及脑脊液中特定蛋白的变化,获得反应疾病的发生、进展、以及机体对疾病产生的代偿机制如神经保护、组织重塑、炎症反应等信息。蛋白质组(Proteome)概念的内容主要包括对全蛋白质的定性、定量和从功能角度分析大量蛋白质,这是一个整体的、动态的概念。蛋白质组学(Proteomics)是指根据蛋白质种类、数量、局部存在的时间、空间上的变化来研究表达于细胞、组织及个体中的全部蛋白质(proteome),并从其结构和功能的角度综合分析生命活动的一门科学。蛋白质组学研究最显著的特点是能对生理与病理状态下的体液、组织或细胞中蛋白质组分进行大通量的综合分析,这有助于对复杂多变的神经系统疾病进行较为全面的认识。本实验即对MS及NMO患者的血清和脑脊液进行蛋白质组学研究,进行蛋白分离、蛋白鉴定,分析探讨MS、NMO各自的血清和脑脊液蛋白组学特点以及行两者之间的比较研究,以期深入探讨两者的病理机制,并对MS、NMO的诊断及两者的鉴别诊断提供一定的辅助依据。第一部分MS患者血清和脑脊液的蛋白质组学研究目的：建立MS血清和脑脊液2D电泳图谱,分别观察MS与正常对照组血清和脑脊液2D电泳的表达差异；对差异表达的蛋白进行鉴定,并探讨其意义。方法：1.采集MS组和正常对照组血清和脑脊液,进行全蛋白提取、Bradford法蛋白定量。2.分别将血清和脑脊液样品均等份混合后重复跑2D电泳三遍,第一向为等电凝聚凝胶电泳,第二向为SDS聚丙烯酰胺凝胶电泳。3.硝酸银染法染后,并应用ImageMaster 2D Platinum5.0软件对图像进行分析。4.分别比较血清和脑脊液结果,选择两组间感兴趣的蛋白点(两组2D电泳图谱中点体积百分比差异两倍以上或者具有有无差异的点)切胶取样、酶解消化,以高敏度HD-ms/ms质谱分析进行蛋白鉴定。5.利用MASCOT在线软件进行检索蛋白质。结果：(一)血清结果1.MS组血清2D电泳凝胶图谱共获得414个蛋白点,正常对照组血清2D电泳凝胶图谱共获得476个蛋白点。2.MS组与正常对照组血清2D电泳凝胶图谱相比,匹配蛋白点为345个MS组相比正常组表达上调的蛋白点159个,其中上调差异大于2倍以上的蛋白点42个,表达下调的蛋白点186个。两组未匹配的蛋白点,MS组69个,正常对照组131个。3.MS组血清2D电泳凝胶图谱与正常对照组相比未表达的蛋白点共81个4.通过HD-ms/ms质谱分析和MASCOT网络检索,鉴定两个差异蛋白质：免疫球蛋白λ和角蛋白83,前者仅见于多发性硬化血清2D凝胶图谱,后者在MS组中的表达比正常对照组差异远大于2倍。(二)脑脊液结果1.MS组CSF 2D电泳凝胶图谱共获得118个蛋白点,正常对照组CSF的2D电泳凝胶图谱共获得350个蛋白点。2.MS组与正常对照组CSF 2D电泳凝胶图谱相比,匹配蛋白点为51个,MS组相比正常组表达上调的蛋白点38个,其中上调差异大于2倍以上的蛋白点24个,表达下调的蛋白点13个。两组未匹配的蛋白点,MS组67个,正常对照组299个。3.通过HD-ms/ms质谱分析和MASCOT网络检索,鉴定三个差异蛋白质：前白蛋白、角蛋白1和角蛋白9,前白蛋白仅见于多发性硬化CSF的2D凝胶图谱,角蛋白1在MS组无表达而在正常对照组有表达,角蛋白9在MS组中的表达比正常对照组差异远大于2倍。结论：1.多发性硬化患者与正常对照组血清2D凝胶电泳图谱对比有多种蛋白质表达呈显著差异。2.鉴定了两个血清差异表达蛋白免疫球蛋白λ(Ig lamda chain)和角蛋白83(keratin 83)。免疫球蛋白λ高表达可能与MS的免疫机制相关。3.发性硬化患者与正常对照组CSF的2D凝胶电泳图谱对比有多种蛋白质表达呈显著差异。4.鉴定了三个脑脊液差异表达蛋白前白蛋白、角蛋白1和角蛋白9。第二部分NMO患者血清和脑脊液的蛋白质组学研究目的：建立NMO血清和脑脊液2D电泳图谱,分别观察NMO与正常对照组血清和脑脊液2D电泳的表达差异；对差异表达的蛋白进行鉴定,并探讨其意义。方法：1.采集NMO组和正常对照组血清和脑脊液,进行全蛋白提取、Bradford法蛋白定量。2.分别将血清和脑脊液样品均等份混合后重复跑2D电泳三遍,第一向为等电凝聚凝胶电泳,第二向为SDS聚丙烯酰胺凝胶电泳。3.硝酸银染法染后,并应用ImageMaster 2D Platinum5.0软件对图像进行分析。4.分别比较血清和脑脊液结果,选择两组间感兴趣的蛋白点(两组2D电泳图谱中点体积百分比差异两倍以上或者具有有无差异的点)切胶取样、酶解消化,以高敏度HD-ms/ms质谱分析进行蛋白鉴定。5.利用MASCOT在线软件进行检索蛋白质。结果：(一)血清结果1.NMO组血清2D电泳凝胶图谱共获得472个蛋白点,正常对照组血清2D电泳凝胶图谱共获得476个蛋白点。2.NMO组与正常对照组血清2D电泳凝胶图谱相比,匹配蛋白点为374个NMO组相比正常组表达上调的蛋白点175个,其中上调差异大于2倍以上的蛋白点57个,表达下调的蛋白点199个。两组未匹配的蛋白点,NMO组98个,正常对照组102个。3.NMO组血清2D电泳凝胶图谱与正常对照组相比未表达的蛋白点共52个4.通过HD-ms/ms质谱分析和MASCOT网络检索,鉴定一个差异蛋白质：触珠蛋白Hp2,在NMO组中的表达比正常对照组差异远大于2倍。(二)脑脊液结果1.NMO组CSF 2D电泳凝胶图谱共获得155个蛋白点,正常对照组CSF 2D电泳凝胶图谱共获得350个蛋白点。2.NMO组与正常对照组CSF 2D电泳凝胶图谱相比,匹配蛋白点为82个NMO组相比正常组表达上调的蛋白点64个,其中上调差异大于2倍以上的蛋白点47个,表达下调的蛋白点18个。两组未匹配的蛋白点,NMO组73个,正常对照组268个。3.通过HD-ms/ms质谱分析和MASCOT网络检索,鉴定三个差异蛋白质：角蛋白1、角蛋白9和转铁蛋白,三者在NMO组中的表达均比正常对照组差异远大于2倍。结论：1.NMO患者与正常对照组血清2D凝胶电泳图谱对比有多种蛋白质表达呈显著差异。2.鉴定了一个血清差异表达蛋白为触珠蛋白。触珠蛋白的高表达提示其在NMO免疫反应中可能发挥一定免疫调节作用。3.NMO患者与正常对照组CSF的2D电泳图谱对比有多种蛋白质表达呈显著差异。4.鉴定了三个脑脊液差异表达蛋白细胞转铁蛋白、角蛋白9、角蛋白1。5.转铁蛋白的脑脊液高表达可能与NMO患者血脑屏障受损相关,可能是NMO病理过程中BBB受损严重的一个反应指标。第三部分MS与NMO患者血清和脑脊液的蛋白质组学研究目的：分别比较MS与NMO患者血清和脑脊液2D电泳的表达差异；对差异表达的蛋白进行鉴定,并探讨其意义。方法：1.采集MS组与NMO组血清和脑脊液,进行全蛋白提取、Bradford (?)去蛋白定量。2.分别将血清和脑脊液样品均等份混合后重复跑2D电泳三遍,第一向为等电凝聚凝胶电泳,第二向为SDS聚丙烯酰胺凝胶电泳。3.硝酸银染法染后,并应用ImageMaster 2D Platinum5.0软件对图像进行分析。4.分别比较血清和脑脊液结果,选择两组间感兴趣的蛋白点(两组2D电泳图谱中点体积百分比差异两倍以上或者具有有无差异的点)切胶取样、酶解消化,以高敏度HD-ms/ms质谱分析进行蛋白鉴定。5.利用MASCOT在线软件进行检索蛋白质。结果：(一)血清结果1.MS组血清2D电泳凝胶图谱共获得414个蛋白点,NMO组血清2D电泳凝胶图谱共获得472个蛋白点。2.正常组血清有表达且同时在MS组血清有表达而在NMO组血清无表达的蛋白点为52个,正常组血清有表达且同时在NMO组血清有表达而在MS组血清无表达的蛋白点为81个3.通过HD-ms/ms质谱分析和MASCOT网络检索,鉴定二个差异蛋白质：角蛋白83和触珠蛋白Hp2。角蛋白83和触珠蛋白Hp2,两者在MS组和NMO组中均有表达,前者在MS组血清中与NMO组相比表达远大于2倍,后者在NMO组血清中与MS组表达远大于2倍。(二)脑脊液结果1.MS组CSF 2D电泳凝胶图谱共获得118个蛋白点,NMO组CSF 2D电泳凝胶图谱共获得155个蛋白点。2.正常组CSF有表达且同时在NMO组CSF有表达而在MS组CSF无表达的蛋白点为45个,正常组CSF有表达且同时在NMO组CSF无表达而在MS组CSF有表达的蛋白点为14个。3.通过HD-ms/ms质谱分析和MASCOT网络检索,鉴定二个差异蛋白质：前白蛋白和角蛋白1,前白蛋白在MS组CSF中有表达而在NMO组和正常对照组CSF中无表达；角蛋白1在NMO组CSF中有表达而在MS组CSF中无表达。结论：1.MS患者与NMO患者血清、CSF的2D电泳图谱对比有多种蛋白质表达呈显著差异。2.鉴定了四个差异表达蛋白细胞角蛋白83、触珠蛋白Hp2、前白蛋白及角蛋白1,前两者为血清差异表达蛋白,后两者为CSF差异表达蛋白。更多还原

【Abstract】 Background:Both multiple sclerosis (MS) and neuromyelitis optica (NMO) are demyelinating diseases of the central nervous system and their underlying mechanisms are unknown. The primary pathological process is demyelinating change, and the clinical manifestations vary with the involved part of the central nervous system.Although multiple sclerosis and neuromyelitis optica both belong to demyelinating diseases of the central nervous system, there are many differences in the pathogenesis, clinical manifestation, clinical characteristics, treatment protocols, etc between them, particularly after the discovery of autoantibody NMO-IgG in the serum of the NMO patients, the current tendency is that NMO is an isolated disease rather than a subtype of MS. However, the difficulty in the collection of the tissue sample of central nervous system impedes the further investigation of pathological mechanisms of MS and NMO. Moreover, as an important component part of internal environment, the body fluid contains polypeptides, proteins and enzymes which play very important roles in the physiological processes, and the changes of the proteins and polypeptides in quality and quantity can reflect the pathophysiological processes under disease accurately, meanwhile, the complex immune responses in MS and NMO result dynamic changes of the types and quantity of the proteins in cerebrospinal fluid, serum, brain tissue and various subcellularorganelle. In consideration of these factors, the information about the occurrence, progression and the compensatory mechanisms of body to disease (e.g. nerve protection, tissue remodeling, inflammatory reaction etc) may be obtained through the study on the changes of special proteins in serum and cerebrospinal fluids of the patients with MS or NMO.The conception of proteome is an integral and dynamic conception, which mainly includes the qualitative analysis and quantification of total proteins and the functional analysis of massive proteins. Proteomics is a science studying the proteome expressed in cell, tissue and individual on the basis of the changes of the types and quantity of proteins as well as the time the space of local presence and analyzing life activities comprehensively from the structural and functional aspects. The most significant feature of the proteomics study is its capability of high throughput comprehensive analysis to the protein components in body fluid, tissue or cells under physiological and pathological conditions, which contributes to the overall understanding about the complex and treacherous diseases of nervous system.This experiment includes the proteomics study on the serum and cerebrospinal fluids from the patients with MS or NMO, protein isolation and identification, the analysis and discussion about the proteomics characteristics of the serum and cerebrospinal fluids from the patients with MS or NMO as well as the comparative study on these diseases in order to explore the pathological mechanisms of them further and provide some auxiliary evidence for the diagnosis and differential diagnosis of MS and NMO. Part One Proteomics Study On The Serum And Cerebrospinal Fluids From MS PatientsPurpose:To establish the 2-D electrophoresis pattern of the serum and cerebrospinal fluids from MS patients, observe the differential expression through the comparison of the 2-D electrophoresis results of the serum and cerebrospinal fluids between the MS group and normal control group, identify the proteins with differential expression and discuss the significance.Method:1. Collect the samples of serum and cerebrospinal fluids from MS group and the normal control group, conduct total protein isolation and quantify the protein by Bradford method.2. The 2-D electrophoresis of the sample with equal amount of serum and cerebrospinal fluid is run in triplets, in which, the first dimension is isoelectric focusing electrophoresis and the second is SDS-PAGE gel electrophoresis.3. Following silver staining, the software ImageMaster 2D Platinum5.0 is used to analyze the images.4. Select the interested proteins spots of both groups (the difference of the volume percent for the spots in the 2-D electrophoresis patterns of two groups is over twofold or the spots with the difference of presenting or not presenting), remove the spots from the gel for sampling, digest the samples by enzymolysis and identify the proteins by high-sensitivity HD-ms/ms mass spectrometry.5. The online software MASCOT is utilized to search the proteins.Result:Ⅰ. Results of serum: 1. There are 414 protein spots in the 2-D electrophoresis pattern of the serum from MS group and 476 proteins in the 2-D electrophoresis pattern of the serum spots from the normal control group in total.2. Comparison of the 2-D electrophoresis pattern of serum between the MS group and the normal control group showed that there are 345 matched protein spots,159 proteins spots in MS group are up-regulated compared with those in the normal control group, among which, the differences between 42 proteins spots are over twofold, and 186 proteins spots in MS group are down-regulated. For the unmatched proteins spots in these two groups, there are 69 spots in MS group and 131 spots in the normal control group.3. Compared with the 2-D electrophoresis pattern of serum from the normal control group, the number of unexpressed proteins spots in MS group is 81.4. Two differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:immunoglobinλ, and keratin 83, the former only presents in the 2-D electrophoresis pattern of serum from the patients with multiple sclerosis, and the expression of the latter in MS group is far over twofold of that in the normal control group.Ⅱ. Results of CSF:1. There are 118 protein spots in the 2-D electrophoresis pattern of CSF from MS group and 350 proteins in the 2-D electrophoresis pattern of CSF from the normal control group in total.2. Comparison of the 2-D electrophoresis pattern of CSF between the MS group and the normal control group showed that there are 51 matched protein spots,38 proteins spots in MS group are up-regulated compared with those in the normal control group, among which, the differences between 24 proteins spots are over twofold, and 13 proteins spots in MS group are down-regulated. For the unmatched proteins spots in these two groups, there are 67 spots in MS group and 299 spots in the normal control group.3. Three differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:prealbumin, keratinl and keratin 9, prealbumin only presents in the 2-D electrophoresis pattern of CSF from the patients with multiple sclerosis, keratinl only presents in the 2-D electrophoresis pattern of CSF from the normal control group and the expression of keratin 9 in MS group is far over twofold of that in the normal control group.Conclusion:1. The expression of a variety of proteins in the 2-D electrophoresis pattern of serum was significantly different in the Multiple sclerosis patients comparison to normal control group.2. Ig lamda chain and keratin 83 have been identified as significantly different serum proteins. Enhanced expreesion of Ig lamda chain in serum may be related to Immune mechanism of MS.3. The expression of a variety of proteins in the 2-D electrophoresis pattern of CSF was significantly different in the Multiple sclerosis patients comparison to normal control group.4. Prealbumin, keratinl and keratin 9 have been identified as significantly different CSF proteins.Part Two Proteomics Study On The Serum And Cerebrospinal Fluids From NMO PatientsPurpose:To establish the 2-D electrophoresis pattern of the serum and cerebrospinal fluids from NMO patients, observe the differential expression through the comparison of the 2-D electrophoresis results of the serum and cerebrospinal fluids between the NMO group and normal control group, identify the proteins with differential expression and discuss the significance.Method:1. Collect the samples of serum and cerebrospinal fluids from NMO group and the normal control group, conduct total protein isolation and quantify the protein by Bradford method.2. The 2-D electrophoresis of the sample with equal amount of serum and cerebrospinal fluid is run in triplets, in which, the first dimension is isoelectric focusing electrophoresis and the second is SDS-PAGE gel electrophoresis.3. Following silver staining, the software ImageMaster 2D Platinum5.0 is used to analyze the images.4. Select the interested proteins spots of both groups (the difference of the volume percent for the spots in the 2-D electrophoresis patterns of two groups is over twofold or the spots with the difference of presenting or not presenting), remove the spots from the gel for sampling, digest the samples by enzymolysis and identify the proteins by high-sensitivity HD-ms/ms mass spectrometry.5. The online software MASCOT is utilized to search the proteins.Result:Ⅰ. Results of serum:1. There are 472 protein spots in the 2-D electrophoresis pattern of the serum from NMO group and 476 proteins in the 2-D electrophoresis pattern of the serum spots from the normal control group in total.2. Comparison of the 2-D electrophoresis pattern of serum between the NMO group and the normal control group showed that there are 374 matched protein spots, 175 proteins spots in NMO group are up-regulated compared with those in the normal control group, among which, the differences between 57 proteins spots are over twofold, and 199 proteins spots in MS group are down-regulated. For the unmatched proteins spots in these two groups, there are 98 spots in NMO group and 102 spots in the normal control group.3. Compared with the 2-D electrophoresis pattern of serum from the normal control group, the number of unexpressed proteins spots in NMO group is 52.4. One differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:haptoglobin Hp2, and the expression of which in NMO group is far over twofold of that in the normal control group.Ⅱ. Results of CSF:1. There are 155 protein spots in the 2-D electrophoresis pattern of CSF from NMO group and 350 proteins in the 2-D electrophoresis pattern of CSF from the normal control group in total.2. Comparison of the 2-D electrophoresis pattern of CSF between the NMO group and the normal control group showed that there are 82 matched protein spots,64 proteins spots in MS group are up-regulated compared with those in the normal control group, among which, the differences between 47 proteins spots are over twofold, and 18 proteins spots in NMO group are down-regulated. For the unmatched proteins spots in these two groups, there are 73 spots in NMO group and 268 spots in the normal control group.3. Three differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:transferrin, keratin 1 and keratin 9. The expression of transferrin, keratin 1 and keratin 9 in NMO group is far over twofold of that in the normal control group. Conclusion:1. The expression of a variety of proteins in the 2-D electrophoresis pattern of serum was significantly different in the NMO patients comparison to normal control group.2. Haptoglobin Hp2 has been identified as significantly different serum proteins. Enhanced expreesion of haptoglobin Hp2 in serum may be related to Immune regulation mechanism of NMO.3. The expression of a variety of proteins in the 2-D electrophoresis pattern of CSF was significantly different in the NMO patients comparison to normal control group.4. Transferrin, keratinl and keratin 9 have been identified as significantly different CSF proteins.5. Enhanced expreesion of Itransferrin in CSF may be related to blood-brain barrier injury of NMO.Part Three Proteomics Study On The Serum And Cerebrospinal Fluids From MS and NMO PatientsPurpose:To compare the 2-D electrophoresis pattern of the serum and cerebrospinal fluids between MS and NMO patients, identify the proteins with differential expression and discuss the significance.Method:1. Collect the samples of serum and cerebrospinal fluids from MS group and NMO group and the normal control group, conduct total protein isolation and quantify the protein by Bradford method.2. The 2-D electrophoresis of the sample with equal amount of serum and cerebrospinal fluid is run in triplets, in which, the first dimension is isoelectric focusing electrophoresis and the second is SDS-PAGE gel electrophoresis.3. Following silver staining, the software Image Master 2D Platinum5.0 is used to analyze the images.4. Select the interested proteins spots of both groups (the difference of the volume percent for the spots in the 2-D electrophoresis patterns of two groups is over twofold or the spots with the difference of presenting or not presenting), remove the spots from the gel for sampling, digest the samples by enzymolysis and identify the proteins by high-sensitivity HD-ms/ms mass spectrometry.5. The online software MASCOT is utilized to search the proteins.Result:I. Results of serum:1. There are 414 protein spots in the 2-D electrophoresis pattern of the serum from MS group and 472 proteins in the 2-D electrophoresis pattern of the serum spots from NMO group in total.2. While at the same time expressed in the 2-D electrophoresis pattern from normal control group, for the unmatched proteins spots in the following two groups, there are 52 spots in MS group and 81 spots in NMO group.3. Two differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:haptoglobin and keratin 83. The expression of the former in MS group is far over two fold of that in NMO group and the expression of the latter in NMO group is far over twofold of that in MS group.Ⅱ. Results of CSF:1. There are 118 protein spots in the 2-D electrophoresis pattern of CSF from MS group and 155 proteins in the 2-D electrophoresis pattern of CSF from tNMO group in total. 2. While at the same time expressed in the 2-D electrophoresis pattern from normal control group, for the unmatched proteins spots in the following two groups, there are 14 spots in MS group and 45 spots in NMO group.3. TWO differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:prealbumin and keratin 1, prealbumin only presents in the 2-D electrophoresis pattern of CSF from the patients with multiple sclerosis, keratin 1 only presents in the 2-D electrophoresis pattern of CSF from the NMO group.Conclusion:1. The expression of a variety of proteins in the 2-D electrophoresis pattern of serum and CSF was significantly different in the Multiple sclerosis patients comparison to NMO group.2. Haptoglobin, keratin 83, Prealbumin and keratinl have been identified as significantly different proteins.更多还原