本研究利用檸檬酸氧化鐵奈米粒子透過磁力攪拌作用捕捉類澱粉蛋白寡聚體加速其聚集，此現象可促進腦內微膠細胞吞噬能力，進而降低類澱粉蛋白寡聚體對類神經細胞造成的神經毒性及神經發炎反應，用以作為阿茲海默症之治療策略。
此研究中所使用的氧化鐵奈米粒子是將熱裂解法合成的油酸氧化鐵奈米粒子經由配位基交換反應將表面置換成檸檬酸，形成直徑20奈米且可穩定分散於水相之檸檬酸氧化鐵奈米粒子。體外試驗結果顯示，透過外加磁力攪拌器使檸檬酸氧化鐵奈米粒子進行攪拌作用，可捕捉類澱粉蛋白寡聚體集結形成無細胞毒性的磁性斑塊。在體外細胞實驗中可發現，將培養成寡聚體狀態的類澱粉蛋白與細胞共培養後，會造成類神經細胞的細胞突觸退化及細胞凋亡，除此之外也會造成微膠細胞轉變為吞噬能力較差的發炎型態並刺激其分泌細胞發炎激素如：介白質–6和腫瘤壞死因子–α。若在共培養時給予檸檬酸氧化鐵奈米粒子並施加磁力攪拌，使類澱粉蛋白寡聚體聚集形成磁性斑塊，即可大幅降低對類神經細胞的細胞毒性，微膠細胞的發炎激素表現也會被抑制，並促進其對磁性斑塊的吞噬能力。
在小鼠動物實驗中，從巴恩斯迷宮測試可發現相較於只注射類澱粉蛋白寡聚體的組別，將類澱粉蛋白寡聚體與檸檬酸氧化鐵奈米粒子一同注射至小鼠腦部海馬迴區域並施予磁力攪拌後，小鼠可回復正常的空間學習與記憶能力，不論在目標區域的停留時間或是行走路徑相較於未治療組別皆有顯著差異。從小鼠大腦切片組織染色結果可觀察到，施予治療組別的類澱粉蛋白寡聚體會聚集為較大的斑塊，不論是類神經細胞的存活率或微膠細胞、星狀膠細胞的活化情形皆獲得改善。
由上述結果可推論，氧化鐵奈米粒子透過磁力攪拌捕捉類澱粉蛋白寡聚體集結形成磁性斑塊確實可降低對類神經細胞的
毒性，並提升微膠細胞對類澱粉蛋白的吞噬作用，降低類澱粉蛋白對腦部傷害與發炎現象。

The aim of this study is to investigate the neurotoxicity and neuroinflammation in brains caused by Amyloid beta oligomers (oAβ) and using water-dispersed citrate super-paramagnetic iron oxide nanoparticles (CA-SPIONs) to develop a magnetic-stirred induced capture of oAβ into magnetic plaques used as a treatment strategy for Alzheimer’s disease. For this purpose, the oleic acid iron oxide which synthesized by thermal decomposition method were modified the surface by ligand exchange reaction to form citrate iron oxide nanoparticles (CA-SPIONs). The CA-SPIONs were spherical nanoparticles with a diameter of 20 nm and well-dispersed in the physiological environment. Under magnetic stirring, CA-SPIONs led aggregation of amyloid beta to become magnetic plaques and reduced highly cytotoxic oAβ forming. Before the treatment, the neuron-like cells, N2a cells, were incubated with amyloid beta in several pre-incubated times to confirm the cytotoxicity effects of amyloid beta in each form. After CA-SPIONs treated under magnetic stirring, the cytotoxicity caused by oAβ on N2a cells showed to be reduced owing to the capture of oAβ into magnetic plaques. Furthermore, the neuroinflammation induced by cytokines secretion from microglial cells with oAβ showed to be obviously decreased after CA-SPIONs/magnetic stirring treatment, and it also promoted the microglial phagocytotic efficiency of oAβ.
The in vivo study about Aβ induced brain damage and therapeutic effect by CA-SPIONs/magnetic stirring was tested by Barnes maze to demonstrate mouse spatial learning and memory ability. After CA-SPIONs/magnetic stirring treatment, the spatial learning and memory ability of Aβ-induced mice recovered as the control group. Neither neurocytotoxicity nor reactive microglial activation in Aβ-induced mice brain tissues were found to be reduced. These results strongly showed the magnetic stirring treatment with CA-SPIONs developed as a valid Alzheimer’s disease treatment.

目錄 1
圖目錄 5
表目錄 6
摘要 7
Abstract 8
一、研究動機 9
二、文獻回顧 11
1. 阿茲海默症 (Alzheimer’s disease, AD) 11
2. 類澱粉蛋白 (Amyloid beta 1-42, Aβ1-42) 11
3. 神經發炎 (Neuroinflammation) 13
5. 類澱粉蛋白學說治療策略 14
6. 超順磁氧化鐵 (Super-paramagnetic Iron Oxide Nanoparticles, SPIONs) 18
三、研究方法 20
1. 奈米氧化鐵之製備與鑑定 20
1.1. 油酸氧化鐵奈米粒子 (OA-SPIONs) 製備方法 20
1.2. 檸檬酸氧化鐵奈米粒子 (CA-SPIONs) 製備方法 20
1.3. 氧化鐵奈米粒子 (SPIONs) 之性質鑑定 21
1.4. 檸檬酸氧化鐵奈米粒子 (CA-SPIONs)之穩定性 21
1.5. 檸檬酸氧化鐵奈米粒子 (CA-SPIONs) 之攪拌作用 22
2. 類澱粉蛋白 (Aβ1-42) 模型之建構與鑑定 22
2.1. 類澱粉蛋白 (Aβ1-42) 模型製備之前處理 22
2.2. 類澱粉蛋白(Aβ1-42)模型之製備 22
2.3. 類澱粉蛋白 (Aβ1-42) 模型之THT檢測 23
2.4. 類澱粉蛋白 (Aβ1-42) 模型之圓漬點墨法 (Dot blot) 23
2.5. 類澱粉蛋白 (Aβ1-42) 模型之形態鑑定 24
2.6. CA-SPIONs攪拌聚集類澱粉蛋白 (Aβ1-42) 之能力評估 24
2.7. 磁性斑塊鑑定 24
3. 細胞實驗 25
3.1. 細胞株及培養環境 25
3.2. 細胞培養液配製與磷酸鹽緩衝溶液 25
3.2.1. 配製DMEM細胞培養液 (For BV-2) 25
3.2.2. 配製DMEM / DMEM Pyruvate細胞培養液 (For N2a) 26
3.3. 細胞繼代 26
3.4. 細胞計數 27
3.5. 評估CA-SPIONs對類神經細胞 (N2a cells) 細胞存活率影響 27
3.6. 評估類澱粉蛋白寡聚體 (oligomeric Aβ1-42, oAβ1-42) 與類神經元細胞 (N2a cells) 共培養之細胞存活率 28
3.7. 評估CA-SPIONs攪拌作用對N2a cells 與Aβ1-42共培養細胞存活率之影響 28
3.8. 評估CA-SPIONs攪拌作用影響N2a cells吞噬oAβ1-42之效率 29
3.9. 評估CA-SPIONs攪拌作用對N2a cells與oAβ1-42共培養細胞型態之影響 30
3.10. 評估CA-SPIONs攪拌作用對N2a cells與oAβ1-42共培養對神經元蛋白 (Neuronal Protein) 生成之影響 30
3.11. oAβ1-42與CA-SPIONs攪拌作用影響N2a cells ROS之產生 31
3.12. 評估oAβ1-42與CA-SPIONs攪拌作用影響N2a cells caspase 3之分泌 32
3.13. 評估CA-SPIONs攪拌作用影響微膠細胞 (BV-2 cells) 吞噬Aβ1-42之效率 33
3.14. 評估CA-SPIONs攪拌作用影響BV-2 cells釋放細胞激素 34
3.15. 評估細胞激素對N2a cells存活率影響 34
4. 動物實驗 34
4.1. 動物來源 35
4.2. oAβ1-42注射腦部模擬阿茲海默症 (Alzheimer's disease, AD) 之動物模型建構 35
4.3. AD小鼠治療策略 35
4.4. 築巢實驗 (Nest construction) 35
4.4. 巴恩斯迷宮 (Barnes maze) 36
4.5. 動物犧牲與組織包埋 37
4.5. 組織切片 37
4.6. Thioflavine S染色法 37
4.6. Congo red染色法 38
4.7. 尼氏染色法 (Nissl Staining) 39
4.8. 腦組織 IHC染色法 39
4.9. H&E染色法 40
4.10. 普魯士藍 (Prussian blue) 染色法 40
4.11. 小鼠體重 41
5. 數據統計 41
四、結果與討論 41
1. 氧化鐵奈米粒子特性分析 41
1.1. 氧化鐵奈米粒子性質鑑定 41
1.2. 檸檬酸氧化鐵奈米粒子穩定性 43
1.3. 檸檬酸氧化鐵奈米粒子攪拌作用 44
2. 類澱粉蛋白 (Aβ1-42) 模型鑑定 44
2.3. 類澱粉蛋白模型之THT檢測 44
2.4. 類澱粉蛋白模型之圓漬點墨法 (Dot blot) 45
2.5. 類澱粉蛋白模型之形狀鑑定 46
2.6. CA-SPIONs攪拌聚集類澱粉蛋白能力評估 48
2.7. 磁性斑塊鑑定 48
3. 細胞實驗 49
3.1. CA-SPIONs對N2a cells細胞存活率影響 49
3.2. 類澱粉蛋白寡聚體與類神經元細胞共培養之細胞存活率 50
3.3. CA-SPIONs攪拌作用對N2a cells 與Aβ1-42共培養細胞存活率之影響 50
3.4. CA-SPIONs攪拌作用影響N2a cells吞噬oAβ1-42之效率 51
3.5. CA-SPIONs攪拌作用對N2a cells 與oAβ1-42共培養細胞型態之影響 52
3.6. CA-SPIONs攪拌作用對N2a cells與oAβ1-42共培養細胞神經元蛋白生成之影響 53
3.8. oAβ1-42與CA-SPIONs攪拌作用影響類N2a cells caspase 3之分泌 56
3.9. CA-SPIONs攪拌作用影響BV-2 cells吞噬Aβ1-42 57
4. 動物實驗 59
4.1. 築巢實驗 (Nest construction) 59
4.2. 巴恩斯迷宮 (Barnes maze) 61
4.3. Thioflavine S染色 62
4.4. Congo red染色 63
4.5. Aβ1-42 IHC染色 64
4.6. 尼氏染色法 65
4.7. Caspase 3 IHC染色 65
4.8. Iba1 IHC染色 66
4.10. 腦組織H&E染色 68
4.11. 腦組織Prussian blue染色 69
4.12. 小鼠體重 69
五、結論 71
六、參考資料 72

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