臺灣博碩士論文加值系統

細胞與動物實驗指出補充葉酸(folic acid)有助於降低homocysteine，進而減少氧化壓力，增加細胞的存活率。先前實驗室的研究證實葉酸在早期阿茲海默症(Alzheimer’s disease, AD)的進展可以增強AD治療藥物之神經細胞保護效果。Folic acid為食品營養補充劑，folinic acid為臨床治療癌症用藥，此兩種葉酸形式(營養補強與藥物使用)是否能對AD基因轉殖鼠認知功能與腦部病理病變改善，尚不清楚。因此，本研究的目的為探討以基因轉殖triple-transgenic AD (3xTg-AD)小鼠的模式，給予葉酸衍生物對晚期AD神經病變之治療效果。16月齡的40隻3xTg-AD小鼠平均分三組，補充folic acid (FA)組、補充folinic acid (FN)組、未補充對照組control (C)，以管灌餵食的方式給予12 mg/kg/day的兩種葉酸衍生物，餵食 3個月。以微生物乳酸菌檢驗法分析組織葉酸含量，以蘇木紫-伊紅染色觀察細胞存活情形，以免疫組織化學染色檢測類澱粉蛋白(amyloid-beta, Aβ)及磷酸化tau，以酵素免疫分析法檢測Aβ 40&42及磷酸化tau，以Morris water maze測試空間記憶學習能力。結果發現，補充葉酸衍生物並未影響動物的攝食與體重，但補充FA (69%)與FN (86%)組的存活率為對照組(54%)之1.3與1.6倍。補充FA及FN組之血清的葉酸營養狀態顯著高於對照組，FA又顯著高於FN (p<0.05)，而腦部皮質的葉酸含量則只有FA組顯著高於對照組(p<0.05)。在免疫染色部分，補充FA及FN組的Aβ plaques顯著比對照組來得少。AD神經病理學的指標，Aβ40、Aβ42及磷酸化tau在補充FA及FN組之腦部皆顯著低於控制組(p<0.05)。血清葉酸營養狀態與Aβ 40&42病理學指標皆呈現顯著負相關(p<0.05)，皮質營養狀態與病理學指標則只有Aβ40有顯著負相關。但在空間記憶學習部分，在三組中無看到明顯的差異。綜合上述，葉酸補充會增加血清葉酸含量，可能可以降低Aβ的沉積，而對空間記憶能力則無明顯的改善，但葉酸補充的確能增加存活率，因此葉酸補充可能可延緩阿茲海默症之進展。

Cell line and animal model had indicated that folate supplementation could help lower homocysteine levels and decrease oxidative stress. We have previously reported that folic acid potentiated effects of Alzheimer’s disease (AD) drug on neuronal protection in early-stage AD development. Folic acid is a kind of nutrient supplement, and folinic acid is a clinical cancer drug. However, in this study, the treatment of late-stage AD with folate derivatives in 3xTg-AD transgenic mice was investigated. Forty 3xTg-AD mice at 16 month-old were divided into three groups fed with the diet in the absence (Control) or presence of folic acid (FA) and folinic acid (FN) supplements (12 mg/kg/day by force feeding). Spatial memory function was assayed by water-maze test before mice were sacrificed at 19 month-old. The data revealed that FA and FN groups had significantly higher levels of serum folate than the controls group after 3 month supplementation (p<0.05). The AD neuropathological markers of Aβ40, Aβ42 or phosphotau levels were significantly lower in the brains of FA and FN groups as compared with the controls (p<0.05). Although these reduced brain pathological severity by FA or FN supplement were not associated with cognition improvement, survival rates of FA (69%) and FN groups (86%) was 1.6 fold-increased in relative to the controls (54%) at 19 month-old. Taken together, the data demonstrated that FA and FN treatments may alleviate pathological severity of late-stage Alzheimer’s disease associated with increased survival rate of 3xTg-AD mice.

中文摘要…………………………………………………………Ⅰ
英文摘要…………………………………………………………Ⅱ
縮寫表……………………………………………………………Ⅲ
致謝…………………………………………………………………Ⅳ
表目錄……………………………………………………………Ⅷ
圖目錄………………………………………………………………Ⅸ
第一章 文獻回顧…………………………………………………………1
一、阿茲海默症致病機轉……………………………………………1
(一) 類澱粉蛋白之形成…………………………………………2
(二) 類澱粉蛋白所引發粒線體之氧化傷害……………………………3
(三) Tau蛋白與Aβ之關係及對阿茲海默症之影響………………4
二、葉酸與阿茲海默症罹患之風險性……………………………………6
(一) 葉酸單碳代謝………………………………………………………………6
(二) 葉酸與阿茲海默症之相關性………………………………8
三、研究動機與目標……………………………………………………………10
第二章 實驗架構與材料方法……………………………………………12
一、實驗設計………………………………………………………………12
二、實驗材料………………………………………………………………13
(一) 實驗動物……………………………………………………………13
(¬二) 動物飼料…………………………………………………………14
三、實驗方法………………………………………………………………14
(一) 動物飼養……………………………………………………………14
(二) 葉酸製備……………………………………………………………14
(三) 動物腦組織處理…………………………………………………14
(四) 水迷宮試驗…………………………………………………………15
(五) 葉酸含量分析………………………………………………………16
(六) Aβ 40&42酵素免疫分析法………………………………17
(七) 磷酸化及全部tau酵素免疫分析法…………………18
(八) 蘇木紫-伊紅染色…………………………………………………19
(九) Aβ42免疫組織化學染色………………………………………19
(十) 磷酸化tau免疫組織化學染色……………………………20
四、資料統計分析…………………………………………………………21
第三章 結果………………………………………………………………22
第四章 討論………………………………………………………………26
第五章 結論………………………………………………………………31
第六章 參考文獻………………………………………………………45

表目錄
表一、補充餵食葉酸衍生物對於3xTg-AD小鼠體重與器官重量及存活率………………………………………………………………………32
表二、補充餵食葉酸衍生物對於3xTg-AD小鼠大腦皮質與血清葉酸營養之影響…………………………………………………………32

圖目錄
圖一、兩種葉酸衍生物的化學結構與結構式……………………………………………………………………………………………………………………………33
圖二、補充餵食葉酸衍生物後3xTg-AD小鼠的H&E染色變化………………………………………………………………………………………………34
圖三、補充餵食葉酸衍生物對於3xTg-AD小鼠Aβ免疫組織化學染色之響………………………………………………………………………35
圖四、補充餵食葉酸衍生物對於3xTg-AD小鼠海馬迴的磷酸化tau免疫組織化學染色之影響…………………………………36
圖五、補充餵食葉酸衍生物對於3xTg-AD小鼠杏仁核的磷酸化tau免疫組織化學染色之影響…………………………………37
圖六、補充餵食葉酸衍生物對於3xTg-AD小鼠的Aβ40沉積量之影響………………………………………………………………………………38
圖七、補充餵食葉酸衍生物對於3xTg-AD小鼠的Aβ42沉積量之影響………………………………………………………………………………39
圖八、補充餵食葉酸衍生物對於3xTg-AD小鼠的全部tau及磷酸化tau量之影響…………………………………………………………40
圖九、3xTg-AD小鼠大腦皮質葉酸營養狀態與大腦病理學指標間的皮爾森相關性………………………………………………………41
圖十、3xTg-AD小鼠血清葉酸營養狀態與皮質病理學指標之間的皮爾森相關性……………………………………………………………42
圖十一、3xTg-AD小鼠血清葉酸營養狀態與大腦病理學指標之間的皮爾森相關性…………………………………………………………43
圖十二、補充餵食葉酸衍生物對於3xTg-AD小鼠在Morris water maze的逃離潛伏期之變化………………………………44

唐銘圻 (2010) 葉酸補充合併阿茲海默症臨床藥物Memantine對Tg2576小鼠腦部基因之調節與降低類澱粉蛋白誘發神經細胞毒性之機制。私立輔仁大學營養科學系碩士論文
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