臺灣博碩士論文加值系統

目前全世界約有2千5百萬人罹患阿茲海默症（Alzheimer’s disease; AD）。在台灣，阿茲海默症佔所有老年失智症人口約六成。阿茲海默症主要病理特徵為患者腦部可發現神經細胞內之神經糾結（Neurofibrillary tangles; NFTs）與細胞外β類澱粉蛋白（Amyloid beta; Aβ）沉積之老化斑（Senile plaques）。研究發現在阿茲海默症病人的腦部有組織發炎的現象和自由基（Reactive oxygen species; ROS）異常增加。目前，阿茲海默症的致病機轉仍未清楚，只知有多項危險因子如年齡、性別、遺傳、慢性疾病等有關。流行病學研究顯示，當年齡增加也會使體內醣化蛋白終極產物(Advanced glycation end products; AGEs)增加，同時阿茲海默症病人的腦部組織發現有大量的AGEs沉積。另外也發現，糖尿病(Diabetic mellitus; DM)病人同時罹患阿茲海默症的機率有顯著的增加，顯示AGEs在阿茲海默症的形成中可能很重要。為釐清AGEs在阿茲海默症中所扮演之角色，本論文主要探討AGEs對於神經瘤細胞株SH-SY5Y中類澱粉前趨蛋白質（Amyloid precursor protein; APP）調控之影響。SH-SY5Y細胞株分別處理不同濃度之AGEs（0-5 mg/ml），檢測APP mRNA、蛋白質、Aβ和ROS的表現。結果顯示AGEs促進APP基因、蛋白質表現並抑制細胞生長，同時其產物Aβ與細胞中ROS的表現量也隨著AGEs濃度而漸增。進ㄧ步探討AGEs與Aβ對細胞的毒殺作用，AGEs合併Aβ處理細胞相較於單獨處理細胞時，更能刺激ROS生成與細胞毒殺之效果。AGEs對於神經細胞之影響可能經由兩種不同途徑，除經由調控增加APP基因、蛋白質與產物Aβ的表現外，也可與Aβ共同作用造成ROS的增加與細胞毒性增強。本研究推論AGEs可能藉由上述兩條不同的途
徑參與阿茲海默症之形成。

There are approximately 25 million Alzheimer’s disease (AD) patients in the world, and about 60％ of dementia patients were caused by AD in Taiwan. Senile plaques which are accumulated by amyloid beta (Aβ) and neurofibrillary tangles (NFTs) are the major hallmarks in the brain of AD patients. Some evidences have shown that inflammation and reactive oxygen species (ROS) increase in AD patients’ brain. But the pathogenesis of AD is unclear. Some risk factors of AD have been reported that include age, sex, genetics and chronic disease. The epidemiological studies have suggested that the levels of advanced glycation end products (AGEs) are enhanced in patients of diabetes mellitus (DM) and ageing procession. At the same time, the evidences indicated moderately increased risks to develop AD in diabetic patients. AGEs may play a role in the pathogenesis of AD. In this study, we proposed that amyloid precursor protein (APP) expression is regulated by AGEs in SH-SY5Y neuroblastoma cell line. Cells were treated with AGEs (0-5 mg/ml) and the expression of APP mRNA were detected, Aβ and ROS. Our data showed that APP mRNA and protein were up-regulated and cell viability was inhibited by AGEs. In the condition medium, the levels of Aβ were increased after AGEs treatment. Furthermore, the increased levels of ROS and cytotoxicity were dose- and time-dependent after cells treated with AGEs and Aβ. In this study, we suggested that AGEs may participate in AD formation through two different pathways. One involves APP gene and Aβ productions. Another, AGEs increased ROS and caused cell toxicity. This report indicated that AGEs maybe an important risk factor in
the pathogensis of AD.

英文縮寫 ----------------------1
中文摘要 ----------------------2
英文摘要 ----------------------4
壹、導論 ----------------------6
一、阿茲海默症-----------------7
1.疾病發現---------------------7
2.致病假說---------------------7
3.病理機轉---------------------8
4.阿茲海默症與基因-------------10
5.APP和α-、β-和γ-secretase ----11
6.β類澱粉蛋白------------------12
7.阿茲海默症與自由基-----------13
二、醣化蛋白終極產物-----------14
1.醣化蛋白終極產物的形成-------14
2.醣化蛋白終極產物與疾病之關係-15
3.醣化蛋白終極產物與自由基-----16
貳、研究構想-------------------18
參、研究材料與方法-------------19
一、AGEs製備與濃縮-------------19
二、細胞培養-------------------19
三、AGEs處理SH-SY5Y細胞株------20
四、細胞計數-------------------20
五、核醣核酸萃取---------------21
六、核醣核酸之定量-------------22
七、cDNA合成-------------------22
八、聚合酶連鎖反應-------------23
九、即時聚合酶連鎖反應---------23
十、蛋白質萃取-----------------24
十ㄧ、蛋白質濃度測定-----------24
十二、聚丙烯胺膠電泳分析-------25
十三、蛋白質轉漬---------------26
十四、西方墨點法---------------26
十五、Aβ1-42免疫螢光酵素測定---27
十六、自由基測定---------------29
十六、Aβ1-42凝集---------------29
十七、統計分析-----------------30
肆、結果-------------------------------31
ㄧ、AGEs對於神經細胞之影響-------------31
二、AGEs調控APP mRNA之表現-------------31
三、神經細胞APP蛋白質表現受AGEs影響----32
四、AGEs影響Aβ的生成-------------------32
五、AGEs刺激神經細胞產生ROS------------33
六、Aβ與AGEs對細胞內ROS的影響----------33
七、Aβ與AGEs對細胞內的毒殺影響---------34
伍、討論-------------------------------35
一、AGEs調控APP之表現------------------35
二、AGEs刺激細胞ROS生成----------------36
三、AGEs對神經細胞的毒性---------------36
陸、結論-------------------------------38
柒、參考文獻---------------------------39
捌、表---------------------------------45
表一、引子序列---------------------45
玖、圖---------------------------------------46
圖一、顯微鏡下觀察AGEs對SH-SY5Y細胞之影響----46
圖二、顯微鏡下觀察AGEs對SH-SY5Y細胞之影響----47
圖三、AGEs對細胞存活率之影響-----------------48
圖四、AGEs調控APP mRNA之表現-----------------49
圖五、神經細胞APP蛋白質表現受AGEs調控--------50
圖六、AGEs影響Aβ1-42生成---------------------51
圖七、AGEs刺激神經細胞產生ROS----------------52
圖九、AGEs與Aβ對細胞內ROS的影響--------------53
圖十、AGEs與Aβ對神經細胞的毒殺影響-----------54
拾、附圖-----------------------------------------------55
附圖一 阿茲海默症患者腦部病理切片圖，神經糾結與斑塊----55
附圖二 阿茲海默症在Aβ產生與清除之假說------------------56
附圖三 APP經由α、β和γ-secrease修飾切割後的產物---------57
附圖四 醣類與蛋白質經非酵素反應合成AGEs----------------58
附圖五 生物體內經由不同的代謝路徑下所產生不同型態之AGEs
------------------------------------------------59
附圖六 研究架構AGEs會影響APP基因與蛋白質之表現，並刺激
細胞產生ROS，對細胞產生毒性作用-----------------60

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