克—雅病患者脑脊液蛋白质组学分析及朊病毒感染中ADAM10变化的研究

发布时间：2018-01-22 21:21

本文关键词： 克-雅病脑脊液朊病毒 TMT标记差异表达蛋白 ADAM10　出处：《中国疾病预防控制中心》2014年博士论文　论文类型：学位论文

【摘要】：可传播性海绵状脑病(Transmissible spongiform encephalopathy, TSEs)也称为朊病毒病(Prion diseases),是一类可感染人类和多种动物罕见的致死性神经退行性疾病,包括人类的克-雅病(Creutzfeldt-Jakob disease, CJD)、吉斯特曼斯特劳斯综合症(Gerstmann-Straussler-Scheinker syndrome, GSS)、Kuru病、家族性致死性失眠症(Fatal familial insomnia, FFI),以及动物的羊瘙痒病(Scrapie)、牛海绵状脑病(Bovine spongiform encephalopathy, BSE)和鹿的慢性消耗性疾病(Chronic wasting disease, CWD)等,其中人类TSEs以CJD为主,包括散发型、遗传型、医源型及变异型四种形式。本文分为两部分,第一部分我们利用TMT标记法串联质谱技术对我国散发型克-雅病(sporadic CJD, sCJD)患者脑脊液(cerebrospinal fluid, CSF)及非CJD患者脑脊液全蛋白进行了蛋白质组学分析。随后利用鸟枪法串联质谱技术对上述样品进行了1～10kDa范围蛋白多肽的肽组分析。通过这些分析鉴定出的差异表达蛋白不仅有助于sCJD患者CSF中潜在生物标志物的筛选,更为深入研究朊病毒致病过程中CSF微环境蛋白的变化规律提供了科学线索。第二部分我们在分子水平证实了PrP能够与去整合素金属蛋白酶10(ADAM10)发生特异的相互作用,主要与成熟型的ADAM10(M-ADAM10),而不是ADAM10的前体(Pro-ADAM10)。 PrP-ADAM10复合物主要分布于细胞膜上。在朊病毒感染的神经细胞系和感染动物脑组织中内源性ADAM10的含量明显下降,并随潜伏时间的增加而减少。第一部分：克-雅病患者脑脊液蛋白质组学分析为检测sCJD和非CJD患者脑脊液中全蛋白表达差异,利用TMT标记法串联质谱技术对我国39例临床诊断sCJD和52例非CJD患者混合CSF进行了蛋白质组学分析。在sCJD及非CJD患者混合脑脊液中共鉴定出437种潜在蛋白,其中49种蛋白在95%置信区间内。差异分析结果显示在这49种蛋白中,相对非CJD组,临床诊断sCJD组中有12种表达显著上调蛋白,13种表达显著下调蛋白。根据差异表达蛋白进行的通路分析显示sCJD患者CSF中影响最大的通路是补体和凝血级联反应通路。随机选择6种CSF差异蛋白进行Western blot验证,结果表明这些蛋白在sCJD与非CJD组中具有与蛋白质组学分析相似的变化趋势。选择磷酸甘油酸变位酶1(上调)和α[-1-抗胰凝乳蛋白酶(下调)分别进行单样品的(24例CJD和24例非CJD)Western blot验证,结果也证实了与蛋白质组学相同的变化趋势。进一步,为了在sCJD患者脑脊液中搜寻大小为1-10kDa的潜在差异蛋白多肽,将40例临床诊断sCJD、32例非CJD具有痴呆症状及17例非CJD不具有痴呆症状的患者脑脊液样品分别等体积混合,并用弱阳离子交换色谱磁珠(MB-WCX)富集1-10kDa蛋白多肽。经胰酶酶解后,用反相-高效液相色谱-电喷雾-四级杆-质谱鉴定、分析富集后的蛋白多肽。结果显示根据各组鉴定的多肽分析,临床诊断sCJD、非CJD痴呆及非CJD非痴呆患者混合脑脊液中分别鉴定出42、53及47种蛋白。在蛋白种类方面,非CJD痴呆组(76.2%)比非CJD非痴呆组(57.1%)更接近l№床诊断sCJD组。最后,我们发现有9种蛋白质特异性分布于临床诊断sCJD组。这些数据显示CSF的蛋白质组学分析不仅促进了潜在的生物标志物的筛选,而且也可成为理解朊病毒致病过程中CSF微环境中蛋白成分变化的科学线索。第二部分：朊病毒感染中ADAM10变化的研究本研究中我们在体外实验中证实了去整合素金属蛋白酶10(ADAM10)能够剪切重组人朊蛋白。通过免疫共沉淀和间接免疫荧光实验发现了PrPC和ADAM10能够发生相互作用,这种相互作用不仪存在于多种体外培养的神经细胞系中,而且还存在于正常仓鼠和小鼠脑组织中。仅仅成熟的ADAM10(移除前肽域后)分了能够绑定天然的PrPc。在体外培养神经细胞系的膜组分中发现了大量的PrP-ADAM10的复合物。在朊病毒感染的SMB细胞模型中,内源性ADAM10的表达水平,尤其是成熟型ADAM10在膜组分中明显减少。免疫共沉淀实验证实在朊病毒感染的SMB-S15细胞的裂解液中未发现PrP-ADAM10的复合物,间接免疫荧光实验结果显示PrP和ADAM10在细胞膜上不存在共定位。而且,我们证实在263K感染终末期的仓鼠和ME7感染终末期的小鼠脑匀浆中,ADAM10的表达水平明显减少,并且随潜伏时间的增加而减少。我们的结果提供了内源性ADAM10和PrP相互作用的可靠分子基础,并且朊病毒感染过程中ADAM10的明显降低提示ADAM10在朊病毒复制和沉积过程中起到了重要的病理、生理作用。
[Abstract]:Transmissible spongiform encephalopathy (Transmissible spongiform, encephalopathy, TSEs) also known as prion disease (Prion, diseases) is a kind of rare animal and humans can be infected many fatal neurodegenerative diseases, including human Creutzfeldt Jakob disease (Creutzfeldt-Jakob disease CJD), Gist Mans Te Laws syndrome (Gerstmann-Straussler-Scheinker syndrome, GSS). Kuru disease, fatal familial insomnia (Fatal familial, insomnia, FFI) and animal scrapie (Scrapie), bovine spongiform encephalopathy (Bovine spongiform, encephalopathy, BSE) of chronic wasting disease and deer (Chronic wasting disease, CWD), the human TSEs based CJD, including sporadic. The genetic type, medical source type and variant four forms. This paper is divided into two parts, the first part we use the TMT labeling method of tandem mass spectrometry in China sporadic CJD (SP Oradic CJD sCJD (cerebrospinal) in cerebrospinal fluid of patients with fluid, CSF) and cerebrospinal fluid of patients with non CJD total protein by proteomic analysis. Then using shotgun tandem mass spectrometry analysis of peptides was 1 ~ 10kDa proteins in these samples. Through the analysis of the identified differentially expressed proteins not only help screening of compounds as potential biomarkers of sCJD patients in CSF, more provide scientific clues for the change of in-depth study of prion pathogenesis in the microenvironment of CSF protein at the molecular level. In the second part we confirm that PrP can go with integrin metalloproteinase 10 (ADAM10) interaction is specific, mainly with the mature type ADAM10 (M-ADAM10), rather than the precursor of ADAM10 (Pro-ADAM10). PrP-ADAM10 complexes are mainly distributed in the cell membrane. The fabric in endogenous neural cell lines and infection in animal brain prion infection group The content of ADAM10 decreased obviously and decreased with the increase of latent time.
Part one: proteomic analysis of cerebrospinal fluid in patients with kya disease
For the expression of protein sCJD and non CJD patients were detected in cerebrospinal fluid, using the TMT labeling method of tandem mass spectrometry for our clinical diagnosis of 39 cases of sCJD and 52 cases of non CJD patients with mixed CSF performed a proteomic analysis. In sCJD and non CJD patients with mixed cerebrospinal fluid were identified 437 kinds of potential protein, among which 49 proteins in 95% confidence interval. The analysis results showed that the difference of the 49 proteins, relative to non CJD group, there are 12 kinds of protein expression was significantly up-regulated in the clinical diagnosis of sCJD group, 13 downregulated proteins. According to the analysis showed that sCJD pathway protein CSF in patients with the greatest impact pathway is the complement and coagulation cascade differential expression. 6 CSF proteins were randomly selected for Western blot verification, the results show that these proteins with proteomic analysis of the similar trends in sCJD and non CJD group. The choice of phosphoglycerate change The enzyme 1 (upregulation) and alpha [-1- anti chymotrypsin (down regulation) were verified by single sample (24 cases of CJD and 24 cases of non CJD) Western blot, and the results also confirmed the same trend of proteomics.
Further, in order to search in cerebrospinal fluid of patients with sCJD the size of potential differences in protein polypeptide 1-10kDa, the clinical diagnosis of 40 cases of sCJD, 32 cases with non CJD dementia and 17 cases of non CJD does not have the symptoms of dementia patients with cerebrospinal fluid samples were mixed with equal volume, and weak cation exchange chromatography beads (MB-WCX) enrichment of 1-10kDa protein polypeptide. After trypsin digestion, by reversed-phase high-performance liquid chromatography electrospray mass spectrometry identification four rod, polypeptides after enrichment analysis. The results showed that according to the analysis of each peptide identification, clinical diagnosis of sCJD, non CJD and non CJD non dementia patients with dementia mixed cerebrospinal fluid were identified in 42,53 and 47 protein. In proteins, non CJD dementia group (76.2%) than non CJD non dementia group (57.1%) close to l no clinical diagnosis of sCJD group. Finally, we found 9 proteins expressed in the clinical diagnosis of sCJD group. These data The proteomic analysis of CSF not only promotes the screening of potential biomarkers, but also can be a scientific clue to understand the changes of protein components in CSF microenvironment during prion pathogenesis.
The second part: Study on the changes of ADAM10 in prion infection
In our study, in vitro experiments confirmed a disintegrin and metalloproteinase 10 (ADAM10) to shear. Recombinant human prion protein by immunoprecipitation and immunofluorescence assay showed that PrPC and ADAM10 can interact in a variety of neural cell lines in vitro in this interaction not only, but also in normal hamster and mouse brain tissue. Only the mature ADAM10 (before removing the peptide domain) can be found in the compound of a large amount of PrP-ADAM10 membrane component binding natural PrPc. cultured nerve cell lines in vitro. In SMB cell model of prion infection, the expression level of endogenous ADAM10, especially mature ADAM10 significantly reduced in membrane fractions. Experiments showed that PrP-ADAM10 complexes were found in lysates of prion infected SMB-S15 cells in CO immunoprecipitation, indirect immunofluorescence test The results showed that PrP and ADAM10 are co localized in the cell membrane. Moreover, we confirmed 263K infection in end-stage hamsters and ME7 infection in end-stage mouse brain homogenate, the expression level of ADAM10 was significantly reduced, and decreases with the increasing of incubation time. To provide a reliable molecular basis of endogenous ADAM10 and PrP interaction our results, and prion infection process reduced ADAM10 suggesting that ADAM10 plays an important pathological in prion replication and deposition process of physiological effects.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.9

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