Proteomic Analysis on Neuroblastoma Cell Line SH-SY5Y Treated with β–Amyloid

澱粉樣蛋白與人類衰弱疾病有關，包括阿茲海默症 (Alzheimer’s disease, AD) 、杭廷頓氏舞蹈症 (Huntington’s disease)、普利昂 (Prion) 相關的疾病，並且可能與白內障的形成有關。這種與老化相關的神經疾病，例如AD，肇於平常循環性可溶的β-澱粉樣蛋白胜肽 (Aβ)，其構形轉變為β-澱粉樣蛋白纖維，後者分別形成老年斑或是神經纖維糾結。一些神經纖維糾結顯示其成份亦包含過度磷酸化的 tau 蛋白質。細胞外Aβ的累積和細胞內tau 蛋白纏結會刺激一系列不正常的訊號和反應，例如細胞內離子衡定的破壞， 不尋常的訊息傳導和長期的發炎反應，都會逐漸導致神經細胞的死亡。 由於人體腦部切片樣品多是病患死亡之後才能取得，樣品稀少，且此時往往已是病症末期。初期的細胞生理變化往往不容易觀察，然而這也許是闡明這一類神經失調疾病機制的重要關鍵。因此這篇論文以人類神經母細胞瘤細胞株 SH-SY5Y作為研究的系統，透過蛋白質體學的方法來研究細胞外的Aβ對於細胞內蛋白質表現所造成的影響。 我們以Aβ處理了神經母細胞瘤細胞，並分別利用二維電泳 (2D-PAGE)以及逆轉錄-聚合酶鏈反應 (RT-PCR)發現了一些蛋白質與基因表現上的變化。同時也利用了能夠辨認磷酸化酪胺酸 (tyrosine)的抗體，進行免疫轉漬 (immunoblotting)的實驗，偵測到蛋白質磷酸化的現象。另外，透過暫時表現基因的方式，發現SH-SY5Y細胞表現正常人類水晶體蛋白αB或其突變序列 (R120G)時，對於Aβ的忍受力會有所不同。由於阿茲海默症與蛋白質摺疊錯誤息息相關，而水晶體蛋白αB為一種小熱休克蛋白質，具有蛋白質分子保護者活性，因此水晶體蛋白αB可能參與了訊息傳導與細胞骨架的調控。從而水晶體蛋白αB本身的性質及其在阿茲海默症病理上扮演的角色，有必要進行更深入詳盡的研究。Amyloid is associated with debilitating human ailments including prominently Alzheimer's disease (AD), Huntington's disease, Prion-related diseases and possibly cataract formation. A central event in this kind of aging-related neurological diseases such as Alzheimer's disease is the conformational change from normally circulating soluble amyloid beta peptides (Aβ) into amyloid fibrils, in the form of senile plaques and neurofibrillary tangles respectively. Some neurofibrillary tangles were also shown to consist of hyperphosphorylated tau proteins. Extracellular Aβ accumulation and intracellular tau tangling would stimulate a series of abnormal signaling and response such as disruption of intracellular ion homeostasis, irregular signal transduction and long-term inflammatory response, all progressively leading to neuronal cell death of neurons. Because brain dissections are often available only after patients' death, sporadic samples obtained usually belong to the diseased state of a very late stage. Generally it is difficult to observe cellular changes in the early stage, which may play a critical role to unravel the mechanism underlying this group of neurological disorders. In this study we have therefore resorted to using the neuroblastoma cell line SH-SY5Y as a model system to study the effects of extracellular Aβ on the protein expression profiles of neuronal cells by a proteomic approach. We treated neuroblastoma cells with Aβ and found some changes in protein and gene expressions as judged by 2D-PAGE and RT-PCR, respectively. Additionally, some proteins modified by phosphorylation were detected by immunoblotting with antibodies against phosphotyrosine. We also found cells transfected for temporary expression of human αB-crystallin or its mutant, αB-crystallin R120G, showed different tolerance to the toxic effects of Aβ. Since AD is concerned with protein misfolding and αB-crystallin is a small heat-shock protein with chaperone activity, this protein might be involved in signal transduction and cell skeleton regulation. Detailed study of αB-crystallin and its role in the pathogenesis of amyloid formation and Alzheimer's disease is warranted for future studies.目錄…………………………………………………………I 縮寫表………………………………………………………II 中文摘要……………………………………………………III 英文摘要……………………………………………………VI 壹、背景介紹………………………………………………1 貳、研究動機………………………………………………5 參、實驗設計………………………………………………6 肆、材料與方法……………………………………………7 一、實驗材料……………………………………………7 二、實驗方法……………………………………………9 伍、實驗結果與討論………………………………………19 陸、未來展望………………………………………………23 柒、圖表……………………………………………………24 圖1……………………………………………………24 圖2……………………………………………………25 圖3……………………………………………………26 表1……………………………………………………28 圖4……………………………………………………29 圖5……………………………………………………30 表2……………………………………………………31 圖6……………………………………………………32 圖7……………………………………………………33 捌、附錄……………………………………………………34 玖、參考文獻………………………………………………3