臺灣博碩士論文加值系統

貝他類澱粉蛋白是造成阿茲海默症形成的一個主要原因。在阿茲海默症的早期可以發現溶小體有一些不正常的改變，貝他類澱粉蛋白也會對阿茲海默症患者產生一些氧化壓力的傷害。這個實驗是要研究貝他類澱粉蛋白1-40與我們純化出來的大鼠腦中的溶小體 ( 純度60~75倍 ) 之間是否有什麼直接反應。當貝他類澱粉蛋白濃度由10 μM一直提升到100 μM所產生活性氧化物的量是隨著濃度呈現劑量效應，但是卻無法在溶小體上產生顯著的脂質過氧化效應。另一方面，在相同濃度下，50 μM的銅離子所產生的脂質過氧化反應比貝他類澱粉蛋白造成的脂質過氧化有顯著的增加。
不管在有或沒有雙氧水作用的情況下，10 μM或者50 μM的貝他類澱粉蛋白加上銅離子與銅離子本身所造成的溶小體脂質過氧化相比沒有顯著的改變，甚至有些許的下降，這說明了在我們實驗的情況下，銅離子是造成溶小體脂質過氧化的主因，而在in vitro的情況下貝他類澱粉蛋白1-40無法造成大鼠腦中溶小體顯著的脂質過氧化增加。

Accumulation of amyloid beta peptide (Aβ) has been proposed to be one key event in the pathogenesis of AD. Disfunction of lysosomes is the earliest change observed in AD patients, and thought to be caused by oxidative damage induced by Aβ. The present study was designed to examine the direct interaction between Aβ40 and lysosomal membrane by using isolated lysosomes, which were purified from rat brain with 60-75 fold purity. Aβ at concentration of 10 to 100 μM caused an increase in reactive oxygen species (ROS) generation and the stimulatory effect was dose dependent. On the other hand, increased Aβ concentration from 10 to 100 μM failed to show any effect on lipid peroxidation of lysosomes, and Cu2+ at 50 μM caused a significant increase in lipid peroxidation as compared to the control or Aβ treatment.
With or without H2O2, the combination of Cu2+ and Aβ40 at 10 or 50 μM caused a similar or slightly lower degree of lipid peroxidation compared with Cu2+ itself, suggesting that Cu2+ is the major stimulator on lipid peroxidation of lysosomes in our experimental condition. And Aβ40 cannot induce a significant increase of lipid peroxidation on rat brain lysosome in vitro.

中文摘要……………………………………………………………………………1
Abstract……………………………………………………………………………2
緒論…………………………………………………………………………………3
阿茲海默症…………………………………………………………………………3
貝他類澱粉蛋白與阿茲海默症……………………………………………………4
貝他類澱粉蛋白的聚集、毒性、以及氧化壓力與銅離子的關係………………5
阿茲海默症與溶小體………………………………………………………………7
實驗目的……………………………………………………………………………9
材料與方法…………………………………………………………………………10
由鼠腦中分離出溶小體……………………………………………………………10
分離出鼠腦中與溶小體重量相近的胞器…………………………………………10
蛋白質濃度測定……………………………………………………………………12
β-Hexosaminidase 活性測定…………………………………………………… 12
Succinate dehydrogenase 活性測定……………………………………………14
Catalase 活性測定 …………………………………………………………… 15
以貝他類澱粉蛋白及銅離子產生活性氧化物……………………………………16
貝他類澱粉蛋白1-40 ( Aβ40 ) 合成……………………………………………16
貝他類澱粉蛋白1-40 ( Aβ40 ) 前處理…………………………………………17
SDS-PAGE 膠體電泳測定 …………………………………………………………17
Coomassie Brilliant Blue 染色法 …………………………………………19
活性氧化物(ROS) 生成量測定……………………………………………………20
測貝他類澱粉蛋白、銅離子、雙氧水在溶小體產生的脂質過氧化……………21
Malondialdehyde ( MDA )生成量測定 ………………………………………21
統計分析方法………………………………………………………………………22
實驗結果……………………………………………………………………………23
由鼠腦中分離出最純的溶小體……………………………………………………23
分離出鼠腦中與溶小體重量相近的胞器 ………………………………………23
溶小體酵素 ( β-Hexosaminidase ) 活性測定 ……………………………… 24
粒線體酵素 ( Succinate Dehydrogenase ) 活性測定 ………………………25
過氧化體酵素 ( Catalase ) 活性測定 ………………………………………25
選定溶小體fraction ………………………………………………………… 26
貝他類澱粉蛋白1-40經過前處理之後的結構觀察 …………………………… 27
測量活性氧化物的量以及溶小體膜的脂質過氧化………………………………28
測定雙氧水(H2O2)的活性氧化物程度以及使溶小體膜
脂質過氧化的程度…………………………………………………………………28
利用貝他類澱粉蛋白使溶小體膜脂質過氧化……………………………………29
測量貝他類澱粉蛋白與銅離子的比例與活性氧化物生成量的關係……………30
利用貝他類澱粉蛋白和銅離子觀察活性氧化物生成與
溶小體脂質過氧化的情形…………………………………………………………31
降低貝他類澱粉蛋白與銅離子的濃度後，
溶小體脂質過氧化的情形…………………………………………………………31
加入雙氧水與貝他類澱粉蛋白和銅離子一起作用………………………………32
討論…………………………………………………………………………………34
結論…………………………………………………………………………………38
參考資料……………………………………………………………………………39
圖表…………………………………………………………………………………44
圖一、Total activity and specific activity of β-hexosaminidase … 44
圖二、Total activity and specific activity of
succinate dehydrogenase ………………………………………………………45
圖三、Total activity and specific activity of catalase………………46
表一、由鼠腦中純化出溶小體 …………………………………………………47
圖四、前處理過後的Aβ40 的電泳圖………………………………………… 48
圖五、不同濃度雙氧水所產生的活性氧化物程度………………………………49
圖六、不同濃度雙氧水所造成的溶小體脂質過氧化……………………………49
表二、Aβ所生成的活性氧化物程度及對溶小體膜所造成的脂質過氧化 …… 50
圖七、圖八、 Aβ和Cu2+的比例不同所產生的活性氧化物程度……………… 51
圖九、Aβ、Cu2+與Aβ.Cu2+ 的活性氧化物生成測定……………………………52
圖十、10 μM Aβ和Cu2+的活性氧化物生成反應…………………………………53
圖十一、10 μM Aβ和Cu2+對溶小體膜的脂質過氧化反應………………………53
表三、Aβ、Cu2+以及雙氧水在活性氧化物及MDA產生的比較………………… 54
表四、1 mM、5 mM 雙氧水和Aβ、Cu2+ 對溶小體的脂質過氧化實驗…………55
表五、不同濃度的雙氧水以及Aβ和Cu2+ 對溶小體脂質過氧化的影響……… 56
圖十二、Aβ40、雙氧水和Cu2+ 對溶小體脂質過氧化的比較………………… 57
附錄一、縮寫檢索表………………………………………………………………58
附錄二、在膜上的澱粉前驅蛋白…………………………………………………59
附錄三、α-、β-、γ -分泌酶在澱粉前驅蛋白上所切割的位置………………60
附錄四、銅離子與貝他類澱粉蛋白結合時所形成的結合物……………………61

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