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系統識別號 U0026-0812200911251877
論文名稱(中文) 阿茲海默氏症的生物標記
論文名稱(英文) Biological markers of Alzheimer’s disease
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 93
學期 1
出版年 94
研究生(中文) 郭智傑
研究生(英文) Chih-Chieh Kuo
學號 t9691403
學位類別 碩士
語文別 中文
論文頁數 103頁
口試委員 口試委員-林靜茹
召集委員-廖寶琦
指導教授-郭余民
中文關鍵字 質譜儀  二維凝膠電泳  生物標記  神經纖維纏結  類澱粉斑  阿茲海默氏症  生物流體 
英文關鍵字 biological marker  two-dimensional gel electrophoresis  neurofibrillary tangle  biologic fluids  amyloid plaque  Alzheimer’s disease  mass spectrometry 
學科別分類
中文摘要   阿茲海默氏症(Alzheimer’s disease,簡稱阿氏症)是隨著年紀增長而罹患率增高的神經退化性疾病,也是一種最常見的失智症(dementia)。目前在全世界估計約有兩千萬的病患,並而這個數字還在快速增加中。阿氏症最主要的特徵就是在行為上會逐漸喪失記憶力和方向感。另外在死後病理上的研究顯示,病患腦中會有類澱粉斑(amyloid plaques)與神經纖維纏結(neuro-fibrillary tangles)的沉積出現,同時神經元數目也會大量減少。這些行為和型態的改變是現在用來診斷阿氏症最常見的方法。但是腦中的病變與神經細胞的退化早在這些特徵出現之前就已經發生了。為了能更早診斷出阿氏症,尋找一個具有高敏感度且高專一性的生物標記是臨床診療的關鍵。最近有關腦疾病的報告顯示,腦組織中的蛋白質會因疾病而有些許的改變,而且有些蛋白質的改變可以在生物流體(biologic fluids)如血清/血漿(serum/plasma)和腦脊髓液(cerebrospinal fluid)中偵測出來。如果能從生物流體中找到一些高靈敏度和高精確度的生物標記(biological markers)來做為阿氏症的早期診斷依據,將可能是一種既方便又便宜的方法。本實驗裡最主要的目的就是嘗試利用二維凝膠電泳(two-dimensional gel electrophoresis,簡稱2-DE)以及質譜儀(mass spectrometry),來尋找出和阿氏症相關的生物標記。實驗中所採用的血漿樣本分別來至10個確定罹患阿氏症和10個非失智的病人。將這兩組血漿的二維凝膠電泳圖樣經過銀染後,每個血漿樣本約可解析出800個蛋白質點。之後再使用軟體將這些蛋白質點量化比對後發現,在阿氏症中有3個蛋白質點的量有明顯增加,另外10個點則是減少。將這些蛋白質點從電泳膠片中取出,並以液相層析串聯式質譜儀鑑定其身分。最後我利用更大量的檢體以及西方點墨法(Western blot)和酵素結合免疫吸附法(Enzyme-linked immunosorbent assay),進一步去確定其中兩個蛋白質apolipoprotein J和Alpha-1- antitrypsin在血漿中的含量。結果顯示阿氏症的病人血漿中alpha-1-antitrypsin的含量有減少的情況,這和2-DE所得到的結果一樣。然而血漿中apolipoprotein J的濃度在阿氏症和非失智的病人之間並無明顯差異。我推測此結果和2-DE發現不符的原因有可能是2-DE看的只是某一磷酸化的apolipoprotein J或是其同分異構物,而西方點墨法和酵素結合免疫吸附法則觀察到血漿中apolipoprotein J的總量。因此要確定這兩個蛋白質是否可以當作阿氏症的標記,還需進一觀察這些特定同分異構物在血漿中的分佈情形。

英文摘要   Alzheimer’s disease (AD) is an age-related neurodegenerative disorder that is the most common form of dementia. It has been estimated that AD affects approximately 20 million people worldwide and this number is rising quickly. AD is characterized by gradual loss of both of cognitive functions and a deterioration of behavior. The postmortem histopathologic studies of brains from AD patients presence of amyloid plaques and neurofibrillary tangles together with the loss of brain weight. These behavioral characteristics and morphological changes are commonly used in the diagnosis for AD. However, the neurodegenerative process has already progressed before these diagnoses can be made. To diagnose AD in more early stage, a biological marker with high sensitivity and specificity for this disease is urgently needed. Previous reports showed that a minor change of the proteins commonly present at tissue constituent induce the onset of brain disease, and some of these proteins are able to be detected in biologic fluids, i.e. blood (serum/plasma) and cerebrospinal fluid (CSF). In this regard, finding some high sensitivity and high specificity markers from biologic fluids for early diagnosis should be possible with an easy and inexpensive blood or CSF test. In this study, I attempt to search AD biological markers using a combination of two- dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Plasma specimens were obtained from 10 AD patients and 10 non-demented controls. Using silver stain we can find about 800 spots in each 2-DE patterns. After comparing the intensity of spots between AD and non-demented control groups, 3 and 10 spots were found to be significantly up and down regulated, respectively. These spots were excised from the gels and subjected to LC-tandem mass spectrometry for protein identification. Two of these protein , apolipoprotein J and alpha-1-antitrypsin, were further validated in plasma samples using Western blot and ELISA. My results revealed that the levels of alpha-1-antitrypsin in AD were lower than those of controls, confirmed the findings of the 2-DE. However, no difference in the concentration of apolipoprotein J was evident between AD and control. This discrepancy may derive from the resolution power between 2-DE and Western blot or ELISA. Many proteins including apolipoprotein J and alpha-1-antitrypsin, can be resoluted by 2-DE into many different isoforms, while such isoform-specific difference can not be observe by Western blot or ELISA. The disease-related isoform-specific variance requires further investigation.

論文目次 中文摘要…………………………………………………04
英文摘要…………………………………………………06
主目錄……………………………………………………08
圖表目錄…………………………………………………10
藥品試劑來源……………………………………………11
英文縮寫對照表…………………………………………13
中英文對照表……………………………………………15
第一章文獻探討…………………………………………17
第一節阿氏症簡介………………………………………18
第二節蛋白質體學………………………………………22
第二章研究動機與目的…………………………………24
第一節研究動機…………………………………………25
第二節研究目的…………………………………………26
第三章實驗材料與方法…………………………………27
第一節本論文實驗流程…………………………………28
第二節常用藥品配方……………………………………29
第三節二維凝膠電泳……………………………………34
第四節西方點墨法………………………………………44
第五節酵素結合免疫吸附法……………………………47
第四章實驗結果…………………………………………50
第一節二維凝膠電泳分析………………………………51
第二節西方點墨法和酵素結合免疫吸附法的確認……57
第五章討論與結論………………………………………60
第一節討論………………………………………………61
第二節結論………………………………………………67
文獻參考…………………………………………………68
附錄一……………………………………………………73
附錄二……………………………………………………75
附錄三……………………………………………………79
附錄四……………………………………………………91
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