以amyloid-β 聚集為目標的阿茲海默氏症治療策略

Abstract

阿茲海默氏症是最常見的老年癡呆症，主要臨床症狀包括Aβ胜肽聚集而成的細胞外澱粉樣蛋白斑與tau蛋白形成的細胞內神經纖維糾結。Aβ胜肽與tau蛋白的聚集往往是基於β-結構互相堆疊而形成。由於藉由破壞蛋白之間的氫鍵可能抑制蛋白聚集，故可能可以從吲哚、多酚類及其衍生物和中藥材篩選出潛在的Aβ胜肽聚集抑制劑。為了達成以上目的，本實驗利用thioflavin T分析法篩選人工合成之化合物。此外，於細胞層面上將Aβ42胜肽與GFP螢光蛋白的N端融合，用於反映Aβ42聚集之程度，並建立於SH-SY5Y細胞和293細胞中，篩選出Tet-On SH-SY5Y細胞與Tet-On 293細胞。Tet-On 293細胞被用來測試植物萃取物與天然或人工合成化合物，藉由綠色螢光訊號區別可延緩或抑制Aβ42聚集之抑制劑。本實驗使用Tet-On 293阿茲海默氏症細胞模式於高通量分析系統，並結合自動顯微鏡與圖像分析自動測試化合物的有效濃度。本實驗分析出10種植物萃取物與人工合成化合物，能提升Aβ42-GFP綠螢光之訊號，其中NTNU-043、NTNU-057、NTNU-059、NTNU-071、NC009-1以及NC009-2等較具潛力，能增加Hsp27蛋白表現，幫助抑制Aβ42聚集。Alzheimer's disease is the most common form of dementia that is pathologically characterized by the presence of extracellular amyloid plaques formed from Aβ peptide and intracellular neurofibrillary tangles formed from tau protein. Aggregation is often based on the formation of cross-β-structure and may be inhibited by disrupting the hydrogen bonds between sheets. Thus screening for indole, polyphenol derivatives and herbal medicines might find out potential inhibitors of Aβ peptide aggregation. To do this, thioflavin T assay was used to screen the synthetic compounds. Also, Aβ42 was fused to the N-terminus of GFP to couple the aggregation state with the fluorescence of GFP and used to generate Tet-On SH-SY5Y cell and 293 cell clones. Tet-On 293 cells were used to screen herbal extracts and natural or synthetic compounds. Inhibitors that retard or block Aβ42 aggregation can be distinguished by the increasing green fluorescent signal. The effective concentration of the tested herbs and compounds will be determined by using a high content analysis system that combines automated microscopy and automated image analysis in 293 AD model. In this study, 10 herbal extracts and synthetic compounds which could significantly increase green fluorescent signal were identified. Among the 10 compounds examined, NTNU-043, NTNU-057, NTNU-059, NTNU-071, NC009-1 and NC009-2 effectively increased green fluorescent signal and enhanced Hsp27 expression.