近年來國人平均壽命隨著醫療進步而延長，老年人口比例逐漸上升，使得台灣進入高齡化社會。其中，骨質疏鬆為常見的老化疾病，好發於老年人與停經婦女。骨質疏鬆症為一種系統性的骨骼疾病，其病徵為骨質流失，骨組織的微結構變差而造成骨骼脆弱，及骨折危險性提高。當蝕骨細胞(Osteoclast)的骨吸收作用大於成骨細胞(Osteoblast)的骨生成作用時，會導致骨流失而造成骨質疏鬆。因此，若能調節骨吸收與骨形成間的平衡，即為治療骨質疏鬆症的關鍵。一氧化氮(nitric oxide, NO)是一內生性氣體分子，參與骨吸收與骨生成的調節。前人研究中，無論是動物實驗或是臨床試驗，皆證實NO可改善骨質流失的現象。由此可知，NO為一極具潛力的治療藥物。但NO在體內的半衰期僅2~6秒，故須增加給藥頻率，造成在治療上的一大限制。本研究開發以癸酸(capric acid)與十八烷(octadecane)組成的微球載體，裝載NO 前驅物 DETA-NONOate。在室溫下，此微球載體以固態形式存在，當其於體溫的環境下，則呈現熔融態；其中，癸酸會提供氫離子使被釋放的DETA-NONOate產生NO，同時具界面活性劑特性的癸酸亦會將NO氣泡圍繞住以形成micelle，穩定NO的結構，並且提供立體障礙，保護NO不易被蛋白(如血紅素、血漿藍銅蛋白等)作用而被降解，進而延長其半衰期。在細胞實驗上，不僅NO可調控骨吸收與骨生成間的平衡，並且癸酸亦具抑制蝕骨細胞的能力。另外在動物實驗中，微球載體的設計不僅能降低給藥頻率，亦能減緩骨質的流失。因此，本研究所製備的微球載體可延長NO的半衰期，且同時具促進成骨生成與抑制骨吸收的能力，進而改善並治療骨質疏鬆症。

As the elderly population grows, the prevalence of osteoporosis is increasing. Osteoporosis is a systemic skeleton disease which happens on elder people and menopausal women because of bone loss. As the microstructure of bone tissue weakened, it will possibly cause bone fracture, which causes pain, disability, inconvenience, and even indirectly lead to death. Under normal physiological conditions, osteoblasts are responsible for bone formation and osteoclasts remove the old bones, which is a continuous process called bone remodeling. Osteoporosis occurs when bone resorption rate is faster than bone formation rate. Therefore, it is critical to strike a balance between bone resorption and bone formation rate in osteoporosis treatments. Nitric oxide is an endogenous gas molecule, which is reportedly able to modulate bone resorption and formation. Previous animal and human studies demonstrate that nitric oxide has the potential to reverse the estrogen deficiency-induced bone loss. However, due to the short half-life of nitric oxide, the frequency of its administration need to be increased, restricting the application of nitric oxide on osteoporosis treatment. Our study developed a temperature-sensitive composite delivery system, which is comprised of capric acid and octadecane, to carry a nitric oxide donor(DETA-NONOate) for treating osteoporosis. At body temperature, the capric acid/octadecane carrier system undergoes phase transition from solid to liquid, causing the release of DETA-NONOate. DETA-NONOate reacts with proton generated by capric acid and forms nitric oxide bubbles, which further regulate the balance between osteoblast and osteoclast. Meanwhile, capric acid, a surfactant molecule, stabilizes the structure of the generated nitric oxide bubbles and prolongs their half-life. On the other hand, capric acid is able to suppress the differentiation of osteoclasts. Accordingly, this carrier system has the biphasic effects of promoting the function of bone-forming osteoblasts and inhibiting the function of bone-resorbing osteoclasts in vitro. In vivo study, this carrier system not only largely decreases frequency of drug application but also successfully alleviates osteoporosis in vivo.

摘要 I
目錄 III
圖目錄 V
表目錄 VII
第一章 緒論 1
1.1 骨質疏鬆之成因 1
1.2 NO對骨細胞之影響 1
1.3 NO應用於骨質疏鬆治療之潛能 3
1.4 NO療法之限制 3
1.5 研究動機與實驗目的 4
1.6 實驗設計流程圖 5
第二章 實驗材料與方法 7
2.1 MPs特性 7
2.1.1 MPs製備之材料 7
2.1.2 MPs製備之方法 7
2.1.3 MPs型態觀察 8
2.1.4 MPs之熔點測定 8
2.1.5 DETA-NONOate穩定性測試 8
2.1.6 一氧化氮體外釋放之偵測 9
2.1.7 原位micelle生成 9
2.1.8 抗降解活性能力測試 10
2.2 細胞實驗 10
2.2.1 細胞培養 10
2.2.2 材料毒性測試 10
2.2.3 成骨細胞增生與存活分析 11
2.2.4 成骨細胞分化分析 11
2.2.5 蝕骨細胞增生與存活分析 11
2.2.6 蝕骨細胞分化分析 12
2.3 動物實驗 12
2.3.1 動物模型建立與給藥方法 12
2.3.2 骨結構分析 13
第三章 實驗結果與討論 14
3.1 MPs特性 14
3.1.1 MPs之大小與型態 14
3.1.2 MPs之熔點、包覆量與十八烷/癸酸流速比例之關係 14
3.1.3 DETA-NONOate穩定性測試 15
3.1.4 NO體外釋放之偵測 16
3.1.5 原位micelle生成之觀察 16
3.1.6 抗降解活性能力測試 18
3.2 細胞實驗 19
3.2.1 材料毒性測試 19
3.2.2 NO對骨細胞的影響 20
3.3 動物實驗 26
3.3.1 骨結構分析 26
第四章 結論 28
參考文獻 29

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