本研究發展一新穎次微米磁棒治療系統，針對腦內瀰漫性類澱粉蛋白的寡聚體(Amyloid-β oligomers)聚集區域進行治療。透過旋轉磁場調控次微米磁棒對Aβ oligomers聚集處進行磁力攪拌後，能有效地將Aβ聚集區的寡聚體(Aβ oligomers)捕捉形成無毒性的磁性斑塊(magnetic plaques)，進一步抑制Aβ寡聚體進入神經元細胞並避免進入細胞後產生毒性導致其死亡，。本研究開發之次微米磁棒以油酸包覆超順磁奈米氧化鐵粒子(oleic acid-coated superparamagnetic iron oxide nanoparticles, OA-SPIONs)為主要材料，利用月桂酸鈉(lauric acid sodium salt, LA)改善油酸覆蓋的奈米氧化鐵粒子於水相中的分散性。次微米磁棒(TEOS modified magnetic stir bars, T-MSBs)的製備則是利用釹鐵硼磁鐵(neodymium magnet)施予磁力線使奈米氧化鐵粒子排列成線狀後，再加入tetraethyl orthosilicate(TEOS)固定其線性結構。本研究製備出的次微米磁棒長度約為2.9 μm，寬度約為100 nm，由於矽殼層(silica shell)表面具大量的矽醇基(silanol groups)，使其具備良好的生理穩定性(pH 7.4, I = 0.15 M)。體外實驗結果顯示，能透過次微米磁棒產生的磁力攪拌作用可以捕捉0.3-160 μM游離的Aβ oligomers，同時集結形成平均面積7-65 μm2的magnetic plaques。於體外細胞實驗結果證實，藉由次微米磁棒產生的磁力攪拌作用捕捉游離的Aβ oligomers，可有效減少Aβ oligomers對類神經元細胞N2a cells造成的毒性。進一步利用共軛焦顯微鏡、西方墨點法評估次微米磁棒產生的磁力攪拌作用，可有效抑制游離的Aβ oligomers造成類神經元細胞N2a cells神經元突觸退化。另一方面，磁性斑塊可有效減少BV-2微膠細胞釋放對神經細胞有害的發炎因子(TNF-α、IL-1β)，同時改善BV-2微膠細胞對Aβ清除的效率。這些結果顯示，T-MSBs的磁力攪拌作用可有效保護腦部細胞，減少Aβ oligomers進入細胞造成神經細胞的凋亡與免疫細胞發炎的不良現象，並可作為阿茲海默氏症治療的次微米裝置。

Aβ oligomers induced neuronal death and microglial inflammation are the major causes of Alzheimer’s disease. In this regard, a smart magnetic stir bar (MSB)-based therapy system which can congregate the neurotoxic Aβ oligomers into nontoxic magnet-induced plaques and impede the penetration of oligomeric species into neurons for novel therapeutic strategies on Alzheimer’s disease was developed in this work. The functionalized MSB was acquired by a neodymium magnet assisted alignment of lauric acid modified superparamagnetic iron oxide nanoparticles into submicron bars while the surfaces will be concomitantly decorated with tetraethyl orthosilicate (T-MSBs) to maintain the bar-shaped structure and improve the colloidal stability. In vitro data demonstrated the MSB-induced mechanical forces actuated the aggregation of Aβ oligomers and significantly reduced the cytotoxicity by means of restricted penetration of Aβ oligomers into neuronal cells (N2a). In consistence with the well development of axon and dendrite through the inhibition of Aβ oligomers into neuron by immunohistochemical examination, the neurite has also shown sound functionality by Western blotting. The confocal laser scanning microscope images confirmed enhanced uptake of magnet-induced plaques on microglia as compared to free Aβ oligomers, leading to an appreciable clearance of the Aβ oligomers. The impaired release of proinflammatory cytokines from microglia considerably promoted the protection effect of targeted neuronal cells. These results demonstrate the greate potential of the T-MSBs as an advanced Alzheimer’s disease therapeutic submicrondevice.

目錄
Abstract p11
中文摘要 p2
前言 p13
一、研究動機 p13
二、文獻回顧 p16
2-1 阿茲海默氏症(Alzheimer’s disease)介紹 p16
2-2 類澱粉蛋白Aβ p16
2-3 神經性發炎反應(Neuroinflammation) p19
2-4 濤蛋白(Tau protein) p20
2-5 阿茲海默氏症體外模型 p21
2-5-1 類神經元細胞體外模型建立 p21
2-5-2 類澱粉蛋白體外模型建立 p21
2-6 類澱粉蛋白學說治療策略發展 p22
三、實驗方法與步驟 p31
3-1次微米磁棒製備與鑑定 p31
3-1-1油酸氧化鐵奈米粒子(OA-SPIONs)製備 p31
3-1-2月桂酸鈉氧化鐵奈米粒子(LA-SPIONs)製備 p31
3-1-3次微米磁棒製備 p32
3-1-4氧化鐵奈米粒子與次微米磁棒鑑定 p33
3-2次微米磁棒穩定度評估 p34
3-3次微米磁棒磁力攪拌作用評估 p34
3-4類澱粉蛋白Aβ42模型建立與鑑定 p34
3-4-1 類澱粉蛋白模型製備前處理 p34
3-4-2 澱粉蛋白寡聚體模型製備與鑑定 p34
3-4-3 類澱粉蛋白斑塊模型製備與鑑定 p35
3-5次微米磁棒攪拌聚集Aβ42能力評估 p35
3-6 磁性斑塊鑑定 p36
3-7體外細胞實驗 p36
3-7-1細胞來源及適合之培養環境 p36
3-7-2配置細胞培養液 p37
3-7-3細胞繼代 p37
3-7-4 細胞存活率分析 p38
3-7-4-1評估不同濃度次微米磁棒對類神經元細胞(N2a cells)細胞存活率影響p38
3-7-4-2 評估(0，500，1500，2500 rpm)狀態的次微米磁棒對類神經元細胞(N2a cells) 細胞存活率與細胞型態影響 p39
3-7-5 評估類澱粉蛋白寡聚體與類神經元細胞(N2a cells)共培養之細胞存活率p39
3-7-6 評估次微米磁棒、類澱粉蛋白寡聚體、磁性斑塊與類神經元細胞(N2a cells)共培養之細胞存活率 p39
3-7-7 細胞膜破損分析 (LDH assay) p40
3-7-8 N2a cells類神經元細胞突觸長度評估 p42
3-7-9 神經元蛋白(Neuronal Protein)-螢光影像評估 p42
3-7-10神經元蛋白-西方墨點法評估 p43
3-7-10-1西方墨點法樣品製備 p43
3-7-10-2 西方墨點法實驗原理 p44
3-7-10-3膠體電泳製備 p44
3-7-10-4 Bradford法蛋白濃度測定 p45
3-7-10-5 西方墨點法實驗步驟 p45
3-7-11微膠細胞(BV-2 cells)吞噬Aβ42螢光影像評估 p45
3-7-12 評估微膠細胞(BV-2 cells)吞噬Aβ42釋放M1型發炎因子 p46
3-7-13 評估M1型發炎因子對類神經元細胞(N2a cells)存活率影響 p47
3-7-14 數據統計 p47
四、結果與討論 p48
4-1 奈米粒子、次微米磁棒特性分析 p48
4-2 次微米磁棒穩定性評估 p50
4-3 次微米磁棒磁力攪拌能力評估 p51
4-4 次微米磁棒之生物相容性評估 p52
4-5類澱粉蛋白模型建立 p54
4-6 評估磁力攪拌作用捕捉類澱粉蛋白 p55
4-6-1 針對npAβ42進行磁力攪拌 p55
4-6-2 針對oAβ42進行磁力攪拌 p58
4-7 磁性斑塊鑑定 p60
4-7-1 面積最大磁性斑塊螢光影像分析 p60
4-7-2 利用ThT assay評估磁性斑塊beta-sheet結構 p63
4-7-3 磁性斑塊結構分析 p64
4-8 磁力攪拌作用捕捉不同濃度之oAβ42 的效果 p66
4-9評估T-MSBs之磁力攪拌作用捕捉oAβ42對類神經元細胞(N2a cells)的治療
情形 p69
4-9-1 細胞存活率評估 p69
4-9-2 細胞膜完整性評估 p72
4-9-3細胞突觸長度評估 p73
4-9-4 神經元蛋白表現-影像評估 p75
4-9-5 神經元蛋白表現-西方墨點法評估 p77
4-9-5-1 mAβ42與oAβ42比較 p77
4-9-5-2 mAβ42與npAβ42比較 p80
4-10 評估oAβ42、mpAβ42、npAβ42對微膠細胞(BV-2 cells)的影響 p81
4-10-1 吞噬作用評估 p81
4-10-2 評估微膠細胞(BV-2 cells)對oAβ42、mpAβ42、npAβ42 p86
4-11 mpAβ42可有效減少微膠細胞(BV-2 ells)釋放M1型發炎因子導致類神經元細胞(N2a cells)凋亡現象 p87
五、結論 p89
六、參考文獻 p90

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