【作者】 王尔松；

【导师】 江澄川； 周嘉伟； 高翔； 胡杰； 王晨； 周晓平；

【作者基本信息】 复旦大学 ， 神经外科学， 2008， 博士

【摘要】 背景:帕金森病(Parkinson’s disease,PD)是最常见的老年性神经疾病之一,其主要临床特征是静止性震颤、肌强直、运动减少和姿势平衡障碍等。PD的病因目前仍不十分清楚。研究认为PD黑质中多巴胺能神经元的变性坏死与蛋白的异常聚集有关。目前PD的诊断主要依据临床表现,尚无特异性的生物标志物用于诊断和鉴别诊断。因而,PD相关蛋白的研究是近几年的热点之一。但是,在CSF中研究PD相关蛋白的报道罕见,在国内尚未见相关报道。作为外科治疗手段之一的慢性脑深部电刺激(DBS)由于其安全、有效、可调和可逆等优点于1987年以来被广泛用于PD的临床治疗。但是其治疗的机制却不清楚。现在已经知道蛋白质在PD发病机制中起着重要的作用,而DBS应用后是否对脑脊液蛋白产生影响,目前国内外还没有文献报道。目的:本研究拟采用蛋白质组学技术对PD患者脑脊液中蛋白质进行研究,建立蛋白表达谱,了解PD相关蛋白的表达情况,找出PD与正常对照组存在的差异蛋白点以尽可能地发现关键性蛋白,以期对PD的发病机制及诊断提供可能线索和生物标志物;以及通过测定手术损伤后和DBS治疗后脑脊液中各种相关蛋白的变化,以探讨DBS治疗的作用机制;同时通过综合评价DBS治疗前后脑脊液各种相关蛋白的变化与统一帕金森病评定量表(unified Parkinson’s disease rating scale,UPDRS)中运动评分及日常生活能力(activities of daily living,ADL)评分之间的相关性,为DBS疗效的判定提供理论依据和可能的生物学检测指标。方法:本研究收集9例PD患者、8例正常对照者、5例DBS治疗者的脑脊液,采用荧光差异凝胶电泳(DIGE)技术分离并筛选PD和正常对照者以及DBS前后脑脊液中差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/MALDI-TOF-TOF)和电喷雾串联质谱(ESI-MS-MS)技术结合蛋白质数据库对这些差异蛋白质进行鉴定并分析。使用免疫印迹(Western-blot)技术对部分鉴定的蛋白质进行验证。分别在药物“开”和“关”的状态下,术前第一天和开机后第7天对5名DBS治疗者进行统一帕金森病评定量表(UPDRS)中的第Ⅱ部分日常生活能力和第Ⅲ部分运动能力进行评分,分别进行统计学处理同时计算其改善率。结果:5名DBS患者在手术中均出现“微毁损效应”,所有患者DBS后症状改善明显,无论在药物“开”还是“关”状态,其运动评分及日常生活能力评分均显著改善。DIGE方法显示匹配点相对比值选择为＞1.5倍或＜-1.5倍时,PD和正常对照间有20个明显的差异蛋白点,其中11个点在PD中上调,9个点下调;匹配点相对比值选择＞1.2倍或＜-1.2倍时,PD和正常对照间有48个明显差异蛋白点,其中19个蛋白点发生下调,29个蛋白点发生上调。MALDI-TOF/MALDI-TOF-TOF和ESI-MS-MS共鉴定出21个蛋白质,其中3个蛋白为白蛋白的异构体(片段?),5个蛋白为免疫球蛋白的片断,2个蛋白为apoA-I异构体,3个蛋白为未知蛋白,13个蛋白属于潜在的PD相关蛋白。其中蛋白MYPT1首次发现在CSF中表达。蛋白apoA-I、MYPT1、tetranectin、C4A首次被发现与PD有潜在相关性。用Western-blot方法验证了apoA-I、MYPT1、C4、IgA、tetranectin、EC-SOD等6个蛋白,发现MYPT1无法测出,其余5个蛋白用Western-blot方法在PD患者和正常对照者CSF中均测出有明确表达,而且在正常对照者和PD患者中的表达与DIGE方法所测均基本一致。但在DBS前后CSF中的表达与DIGE方法所测略有出入。结论:在13个属于PD潜在的相关蛋白中,部分蛋白有可能成为PD的生物标记物。而部分DBS前后改变明显的蛋白,有可能成为监测DBS疗效的生物学指标。DBS治疗的机制之一可能是通过与毁损不完全相同的机制维持或进一步促进微毁损的治疗效果。更多还原

【Abstract】 Background:Parkinson’s disease（PD） is one of the common neurodegenerative disorders characterized by a progressive loss of dopaminergic neurons in the substantia nigra,striatum and putamen,resulting in extrapyramidal motor dysfunction, including resting tremor,rigidity,bradykinesia and postural instability.The pathogenesis of PD is not yet clear.Increasing evidence has suggested that degeneration and loss of dopaminergic neurons in the substantia nigra have links with abnormal protein aggregation.The clinical diagnosis of PD is based entirely on clinical features.There is no special biomarker for PD in diagnosis and differential diagnosis.PD related proteins are becoming research hotspot in recent years. However,the reports on PD related proteins in cerebrospinal fluid（CSF） to date are rare,and there is no report yet on this field in China.As one of the surgical method for PD therapy,deep brain stimulation（DBS） is safe,effective,adaptable,and reversible,and increasingly being used across the world since 1987.However,its primary mechanisms of action are yet poorly understood.It has been known that proteins play important roles in pathological processes of PD.Up to now,there is still no research to report whether DBS has effects on proteins in CSF.Objectives:To compare protein profiles in the CSF between PD patients and control subjects and to identify the PD-related proteins that might serve as potential biomarkers or clues for diagnosis or pathogenesis.Moreover,to quantitate the variation of protein expression in CSF pre-DBS and post-DBS to explore the mechanisms of DBS for the treatment of PD.Evaluating possible relations between the variations of protein expressions in CSF pre- and post-DBS and the Unified Parkinson’s Disease Rating Scale（UPDRS） motor scores and activities of daily living （ADL） scores to find out potential biological markers for monitoring the efficacy of DBS.Methods:The CSF from 9 PD patients,8 control subjects,and 5 DBS patents was studied for the differentially expressed proteins in patients with PD compared with controls,in pre-DBS patients compared with post-DBS by two-dimensional difference gel electrophoresis（2D DIGE） technique,in combination with matrix-assisted laser desorption/ ionization- time of flight（MALDI-TOF） and tandem（TOF-TOF） mass spectrometry or electrospray ionization tandem mass spectrometry（ESI-MS- MS）. Western-blotting was used to validate the data of proteomic analysis.The motor scores and ADL scores of UPDRS for 5 DBS patients were valuated on the day before operation and the 7^th day after chronic stimulation starting（stimulator on） in state of ON medication and OFF medication respectively.Statistic analysis on UPDRS scores were performed for improvement.Results:Micro-lesioning effects were observed during operation in all 5 patients with PD.The scores of motor and ADL were significantly improved after DBS in both on-medication and off-medication condition.DIGE analysis showed that the levels of 20 protein spots were significantly altered in PD CSF when average ratio of the matched spot was assigned as＞1.5 or＜-1.5.Of which,11 spots were up-regulated and 9 spots were down-regulated.48 spots were significantly altered in PD patients compared with controls when average ratio＞1.2 or＜-1.2.Of which 19 spots were considerably down-regulated in the CSF of PD patients,whereas 29 spots were considerably up-regulated.21 unique proteins（including redundancies due to proteolysis and post-translationally modified isoforms） were identified after MS and protein database interrogation.Of them 3 were albumin isoforms（fragments ?）,5 were fragments of immune globin,2 were apolipoproteinA-Ⅰ（apo A-Ⅰ） isoforms,3 were unnamed proteins.13 of 21 proteins were potentially PD-related proteins. Myosin phosphatase target subunit 1（MYPT1） was identified for the first time in CSF, and apoA-Ⅰ,MYPT1,tetranectin,C4A were identified for the first time in the CSF of PD patients.The expression levels of C4,apoA-Ⅰ,IgA,tetranectin,extracellular superoxide dismutase（EC-SOD）,detected by Western-blotting,correlated well with the DIGE results in PD patients and controls,and differed partially from the DIGE results in pre- and post-DBS patients.Conclusions:some of 13 proteins which belonged to potential PD related protein may serve as possible biomarkers for PD,and some of the proteins altered significantly pre- and post-DBS may be useful for better understanding of mechansism of DBS. The role of DBS for PD is probably to sustain or promote effects of micro-lesioning by the mechanism different from lesioning.更多还原