本文關(guān)鍵詞： 克-雅病 腦脊液 朊病毒 TMT標(biāo)記 差異表達(dá)蛋白 ADAM10　出處：《中國(guó)疾病預(yù)防控制中心》2014年博士論文　論文類型：學(xué)位論文

【摘要】：可傳播性海綿狀腦病(Transmissible spongiform encephalopathy, TSEs)也稱為朊病毒病(Prion diseases),是一類可感染人類和多種動(dòng)物罕見(jiàn)的致死性神經(jīng)退行性疾病,包括人類的克-雅病(Creutzfeldt-Jakob disease, CJD)、吉斯特曼斯特勞斯綜合癥(Gerstmann-Straussler-Scheinker syndrome, GSS)、Kuru病、家族性致死性失眠癥(Fatal familial insomnia, FFI),以及動(dòng)物的羊瘙癢病(Scrapie)、牛海綿狀腦病(Bovine spongiform encephalopathy, BSE)和鹿的慢性消耗性疾病(Chronic wasting disease, CWD)等,其中人類TSEs以CJD為主,包括散發(fā)型、遺傳型、醫(yī)源型及變異型四種形式。本文分為兩部分,第一部分我們利用TMT標(biāo)記法串聯(lián)質(zhì)譜技術(shù)對(duì)我國(guó)散發(fā)型克-雅病(sporadic CJD, sCJD)患者腦脊液(cerebrospinal fluid, CSF)及非CJD患者腦脊液全蛋白進(jìn)行了蛋白質(zhì)組學(xué)分析。隨后利用鳥(niǎo)槍法串聯(lián)質(zhì)譜技術(shù)對(duì)上述樣品進(jìn)行了1～10kDa范圍蛋白多肽的肽組分析。通過(guò)這些分析鑒定出的差異表達(dá)蛋白不僅有助于sCJD患者CSF中潛在生物標(biāo)志物的篩選,更為深入研究朊病毒致病過(guò)程中CSF微環(huán)境蛋白的變化規(guī)律提供了科學(xué)線索。第二部分我們?cè)诜肿铀阶C實(shí)了PrP能夠與去整合素金屬蛋白酶10(ADAM10)發(fā)生特異的相互作用,主要與成熟型的ADAM10(M-ADAM10),而不是ADAM10的前體(Pro-ADAM10)。 PrP-ADAM10復(fù)合物主要分布于細(xì)胞膜上。在朊病毒感染的神經(jīng)細(xì)胞系和感染動(dòng)物腦組織中內(nèi)源性ADAM10的含量明顯下降,并隨潛伏時(shí)間的增加而減少。 第一部分：克-雅病患者腦脊液蛋白質(zhì)組學(xué)分析 為檢測(cè)sCJD和非CJD患者腦脊液中全蛋白表達(dá)差異,利用TMT標(biāo)記法串聯(lián)質(zhì)譜技術(shù)對(duì)我國(guó)39例臨床診斷sCJD和52例非CJD患者混合CSF進(jìn)行了蛋白質(zhì)組學(xué)分析。在sCJD及非CJD患者混合腦脊液中共鑒定出437種潛在蛋白,其中49種蛋白在95%置信區(qū)間內(nèi)。差異分析結(jié)果顯示在這49種蛋白中,相對(duì)非CJD組,臨床診斷sCJD組中有12種表達(dá)顯著上調(diào)蛋白,13種表達(dá)顯著下調(diào)蛋白。根據(jù)差異表達(dá)蛋白進(jìn)行的通路分析顯示sCJD患者CSF中影響最大的通路是補(bǔ)體和凝血級(jí)聯(lián)反應(yīng)通路。隨機(jī)選擇6種CSF差異蛋白進(jìn)行Western blot驗(yàn)證,結(jié)果表明這些蛋白在sCJD與非CJD組中具有與蛋白質(zhì)組學(xué)分析相似的變化趨勢(shì)。選擇磷酸甘油酸變位酶1(上調(diào))和α[-1-抗胰凝乳蛋白酶(下調(diào))分別進(jìn)行單樣品的(24例CJD和24例非CJD)Western blot驗(yàn)證,結(jié)果也證實(shí)了與蛋白質(zhì)組學(xué)相同的變化趨勢(shì)。 進(jìn)一步,為了在sCJD患者腦脊液中搜尋大小為1-10kDa的潛在差異蛋白多肽,將40例臨床診斷sCJD、32例非CJD具有癡呆癥狀及17例非CJD不具有癡呆癥狀的患者腦脊液樣品分別等體積混合,并用弱陽(yáng)離子交換色譜磁珠(MB-WCX)富集1-10kDa蛋白多肽。經(jīng)胰酶酶解后,用反相-高效液相色譜-電噴霧-四級(jí)桿-質(zhì)譜鑒定、分析富集后的蛋白多肽。結(jié)果顯示根據(jù)各組鑒定的多肽分析,臨床診斷sCJD、非CJD癡呆及非CJD非癡呆患者混合腦脊液中分別鑒定出42、53及47種蛋白。在蛋白種類方面,非CJD癡呆組(76.2%)比非CJD非癡呆組(57.1%)更接近l№床診斷sCJD組。最后,我們發(fā)現(xiàn)有9種蛋白質(zhì)特異性分布于臨床診斷sCJD組。這些數(shù)據(jù)顯示CSF的蛋白質(zhì)組學(xué)分析不僅促進(jìn)了潛在的生物標(biāo)志物的篩選,而且也可成為理解朊病毒致病過(guò)程中CSF微環(huán)境中蛋白成分變化的科學(xué)線索。 第二部分：朊病毒感染中ADAM10變化的研究 本研究中我們?cè)隗w外實(shí)驗(yàn)中證實(shí)了去整合素金屬蛋白酶10(ADAM10)能夠剪切重組人朊蛋白。通過(guò)免疫共沉淀和間接免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)了PrPC和ADAM10能夠發(fā)生相互作用,這種相互作用不儀存在于多種體外培養(yǎng)的神經(jīng)細(xì)胞系中,而且還存在于正常倉(cāng)鼠和小鼠腦組織中。僅僅成熟的ADAM10(移除前肽域后)分了能夠綁定天然的PrPc。在體外培養(yǎng)神經(jīng)細(xì)胞系的膜組分中發(fā)現(xiàn)了大量的PrP-ADAM10的復(fù)合物。在朊病毒感染的SMB細(xì)胞模型中,內(nèi)源性ADAM10的表達(dá)水平,尤其是成熟型ADAM10在膜組分中明顯減少。免疫共沉淀實(shí)驗(yàn)證實(shí)在朊病毒感染的SMB-S15細(xì)胞的裂解液中未發(fā)現(xiàn)PrP-ADAM10的復(fù)合物,間接免疫熒光實(shí)驗(yàn)結(jié)果顯示PrP和ADAM10在細(xì)胞膜上不存在共定位。而且,我們證實(shí)在263K感染終末期的倉(cāng)鼠和ME7感染終末期的小鼠腦勻漿中,ADAM10的表達(dá)水平明顯減少,并且隨潛伏時(shí)間的增加而減少。我們的結(jié)果提供了內(nèi)源性ADAM10和PrP相互作用的可靠分子基礎(chǔ),并且朊病毒感染過(guò)程中ADAM10的明顯降低提示ADAM10在朊病毒復(fù)制和沉積過(guò)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.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 石琦;高晨;陳操;周偉;張寶云;田嬋;韓俊;董小平;;2009年中國(guó)克雅氏病監(jiān)測(cè)病例特征分析[J];疾病監(jiān)測(cè);2010年10期

2 宋宏新,程軍;瘋牛病(BSE)的檢測(cè)、預(yù)防及控制[J];中國(guó)人獸共患病雜志;2005年03期

3 ;Analysis of the urinary peptidome associated with Helicobacter pylori infection[J];World Journal of Gastroenterology;2011年05期

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本文編號(hào)：1455758