cns脱髓鞘疾病生物标记物的蛋白质组学研究.pdf
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1、硕士学位论文CNS脱髓鞘疾病生物标记物的蛋白质组学研究山东大学硕士学位论文CNS脱髓鞘疾病生物标记物的蛋白质组学研究 摘 要CNS 脱髓鞘疾病(Central nervous system demyelinative diseases)是以 中枢神经系统炎性、多灶性脱髓鞘为主的自身免疫系统疾病,以反复发作,多次 缓解、复发为特征。病灶以主要累及大脑、脑干、小脑为主的称为多发性硬化(Multiple sclerosis,MS)。以主要累及视神经和脊髓为主的称为视神经脊髓炎(Neuromyelitis optica,NMO)。MS是涉及到神经系统的最普通的自身免疫性疾病,是一种影响神经系统传 导且
2、导致进行性神经功能异常的慢性疾病。MS的病理学标志是脑和脊髓的脱髓 鞘斑的发现,并伴有神经系统血管周围炎性细胞浸润、脱髓鞘、星形神经胶质增 生以及相应的轴突损伤。这些症状主要是由于轴突脱髓鞘,从而抑制或阻碍传导 造成的。局部髓鞘再生和炎症的消退有助于症状的改善。NMO,也称Devic,s综合症,是一种感染视神经和脊髓的炎性脱髓鞘疾病,它不同于MS,没有侵及到大脑,视神经和视交叉为本病最多见的病变部位,偶可 累及视束。NMO是一种可以致残以至危及生命的疾病。预后较差,一半以上的病 人在病情发作5年内将发展为双侧视力受损甚至至少一眼完全失明,亦或失去行 走能力。CNS脱髓鞘疾病严重危害人体健康,至
3、今仍没有特异性的生物标记物对其诊 断治疗。脑脊液(cerebrospinal f luid,CSF)是存在于脑室及蛛网膜下腔内的一种无 色透明液体,含有一些小分子物质如盐和肽,蛋白质,酶等。这些物质的变化包 括浓度、蛋白和肽的修饰等都能够精确反映神经系统疾病的病理过程。CSF的异 常变化是CNS脱髓鞘疾病的特点之一。蛋白质组学(proteomics)是指一个细胞或 一个组织/体液基因组所表达的全部蛋白质。蛋白质在生命活动中发挥了重大的 作用,蛋白质组学研究可以为疾病的发病机制已及生物标记物的发现提供一定启 1山东大学硕士学位论文示。CSF蛋白质组学是研究CSF中蛋白质表达的一项有效的技术手段,
4、为研究CNS 疾病的病理生理机制提供了重要信息。本研究以蛋白质组学技术为平台,通过双向电泳分离技术、质谱鉴定技术、ELISA、Western blot验证实验以及蛋白质网络图谱的建立,比较CNS脱髓鞘疾 病(多发性硬化和视神经脊髓炎)组脑脊液与对照组(其他神经系统疾病)脑脊液 的蛋白质组的变化,寻找与该疾病的发生机制相关的生物标记物,以期从蛋白质 及分子水平探讨其发病的机理,为临床诊断、预后及治疗提供有力的理论依据。目的:通过对CNS脱髓鞘疾病患者脑脊液进行比较蛋白质组学研究及生物信 号网络分析,探索与发病机制相关的潜在生物标记物以及生物标记物与该疾病间 的关系,为其发病机理的探索、临床诊断和
5、治疗提供依据。方法:分别收集CNS脱髓鞘疾病组和对照组脑脊液,冰丙酮沉淀法提取总蛋 白,进行双向凝胶电泳分离蛋白质组,Imag eMaster 2D图像分析软件筛选出表 达有显著差异的蛋白质点,应用基质辅助激光解吸电离/飞行时间质谱(MALDI-TOF MS)得到肽质量指纹图谱(PMF),搜索SWISSPROT数据库对其进行鉴 定。以表达显著的蛋白质点为研究目标,从中挑选个别蛋白进行ELISA、Western blot定量和半定量验证,MetaCore,PathwayStudio软件对表达显著的蛋白质进 行相关分析。结果:分别获得MS患者及对照组脑脊液蛋白质组表达图谱,8个蛋白质点 在两组凝胶
6、图谱上存在明显差异,4个点在MS组显著上调,4个点下调,其中 CystatinC 的 ELISA 结果为:(MS 组 4.36 1.22mg/L;对照组 6.00L 68mg/L,P 0.01)o对表达显著的蛋白质成功构建了 MetaCore信号网络图谱。NMO患者及对照组脑脊液蛋白质组表达图谱中,13个蛋白质点在两组凝胶图 谱上存在明显差异,4个点在NM0组显著上调,9个点下调,对Vitamin D binding protein(DBP)进行了 Western blot半定量验证。蛋白质网络图谱表达了蛋白质 点之间的生物学关系。结论:蛋白质的差异表达及其生物信号网络研究有助于在分子水平上对
7、CNS 脱髓鞘疾病的发病机理、鉴别诊断及药物靶点间的关系探索方面提供一定依据,这些疾病相关蛋白可以作为CNS脱髓鞘疾病潜在的生物标记物。关键词:中枢神经系统脱髓鞘疾病;多发性硬化;视神经脊髓炎;蛋白质组 学;脑脊液;生物标记物2山东大学硕士学位论文Proteome analysis of biomarkers in the cerebrospinal fluid of central nervous system demyelinative diseases ABSTRACTCNS demyelinative diseases are autoimmune diseases of the ce
8、ntral nervous system(CNS)with the inf lamation and mulif ocality demyelination.They are characterized by recurrent attack s and relapsing-remitting.In multiple sclerosis(MS),the demyelination is primarily involved in the cerebrum,brainstem and cerebellum.However,in neuromyelitis optica(NMO),the demy
9、elination predominantly af f ects optic nerves and spinal cord.MS,the most common autoimmune disease involving the CNS,is a chronic illness af f ecting CNS pathways and leading to prog ressive neurolog ical dysf unction.The patholog ical hallmark of MS is the demyelinated plaque f ound throug hout t
10、he brain and spinal cord,with CNS perivascular inf iltration of inf lammatory cells,demyelination,astrog liosis,and axonal injury.Symptoms are believed to result primarily f rom axonal demyelination that inhibits or block s conduction.Amelioration of symptoms has been attributed to both partial remy
11、elination and resolution of inf lammation.NMO,also termed Devic s syndrome,is an idiopathic inf lammatory demyelinating disease of the CNS predominantly af f ecting optic nerves and spinal cord,but unlik e MS,does not af iect the brain.The common af f ections are optic nerves and optic chiasma,somet
12、imes involves the optic tract.It is a f requently3山东大学硕士学位论文 disabling,occasionally lif e-threatening disease.Furthermore,the prog nosis of NMO is of ten poor and more than half of patients will develop severe bilateral visual impairment and even visual loss in at least one eye and/or inability to a
13、mbulate without assistance within 5 years of disease onset.CNS demyelinative diseases severely af f ect human health.Up to now,the diag nosis of them are challeng ing f or lack of specif ic biomark ers.Cerebrospinal f luid(CSF)is a promising source of biomark ers f or disorders of the central nervou
14、s system,since the CSF compartment is in close contact with the brain interstitial f luid where biochemical chang es related to the underlying disease are of ten ref lected.Abnormal CSF f inding s are one of the characteristics of CNS demyelinative diseases.Proteomics is the study of identif ying th
15、e entire protein components(proteome)of a cell,tissue or f luid at a g iven point in time.Since proteins play a central role in the lif e of an org anism,the study of proteome of the org anism can provide insig ht into the mechanisms of diseases,which is helpf ul in discovery of biomark ers of the d
16、iseases.Now it is well accepted that CSF proteomics is a powerf ul technique to describe the expression of proteins in CSF.Moreover,it can provide valuable inf ormation about the pathophysiolog ical mechanisms xinderlying the diseases in CNS.In this study,by means of the proteomic analysis with two-
17、dimensional electrophoresis(2-DE),f ollowed by matrix-assisted laser desorption ionization time of f lig ht mass spectrometry(MALDI-TOF MS)and database searching,in combination with the validation of the ELISA,Western blot test,and the establishment of the protein network,we studied the CSF protein
18、alterations beween the CNS demyelinative diseases(MS and NMO)and the control g roups,f ound the candidated biomark ers in CNS demyelinative diseases,expected to investig ated their pathog enesy,diag nosis,prog nosis and treatment on the molecular level.4山东大学硕士学位论文Objective:Comparative proteomics and
19、 biolog ical sig naling network analysis were carried out in the cerebrospinal f luid(CSF)of the patients with CNS demyelinative diseases to investig ate the relations both among the potiental biomark ers and between the biomark ers and the diseases.These candidated biomark ers may be g ive inf orma
20、tion f br studying the pathophysiolog ical process,the clinical diag nosis and treatment of the diseases.Methods:CSF were extracted f rom patients with CNS demyelinative diseases and the controls respectively,and the proteins were precipitated with ice-cold acetone.Then the two-dimensional electroph
21、oresis(2-DE)were applied and the dif f erential expression protein spots were selected with the Imag eMaster 2D-g el sof tware.The peptides mass f ing erprint(PMF)was g ot by the matrix assisted laser desorption ionization time of f lig ht mass spectrometry(MALDI-TOF MS)and searched in SWISSPROT dat
22、abase to identif y dif f erential proteins.The expressed obviously proteins were selected to be conf irmed by subsequent ELISA and Western blot test.Finally,the correlations of these proteins were analized by MetaCore and PathwayStudio sof tware.Results:2-DE maps of CSF with MS and the controls g ro
23、up were g ot.There were eig ht protein spots expressed dif f erentially on the 2-DE maps,in which f our up-reg ulated and f our down-reg ulated protein spots were identif ied in MS.Cystatin C is down-reg ulated and f urther conf irmed by the ELISA assay(4.361.22 mg/L,6.001.68mg/L,P0.01).A map of sig
24、 naling network about these proteins were built by MetaCore integ rated sof tware.By using 2-DE and MALDI-TOF MS we identif ied 13 dif f erent protein spots in NMO g roup.The CSF expression of f our protein spots was enhanced,whereas the expression of nine protein spots was reduced in the NMO g roup
25、 compared with the 5山东大学硕士学位论文control g roup.One of these proteins,vitamin D binding protein(DBP)was conf irmed by the Western blot test(p0.01).Moreover,protein network of 11 proteins was partially g ot,which showed some biolog ical interaction.Conclusions:The dif f erential expressions and biolog i
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- cns 脱髓鞘 疾病 生物 标记 蛋白质 研究
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