臺灣博碩士論文加值系統

腸道中的腸上皮緊密連接具有屏障的功能，而細菌來源的脂多醣會導致腸屏障功能受損，增加發炎性腸道疾病發病的風險，蛋殼膜蛋白表現出潛在的抗發炎活性，卻因其屬於親水性大分子，不易通過腸黏膜，造成吸收阻礙。本研究目的為製備幾丁聚醣/褐藻多醣奈米粒作為口服傳遞載體，包覆可溶性蛋殼膜蛋白以提高其腸道吸收，用於治療腸上皮細胞受損。本實驗成功萃取可溶性蛋殼膜蛋白，並以幾丁聚醣/褐藻多醣奈米粒包覆。當幾丁聚醣、褐藻多醣與可溶性蛋殼膜蛋白重量比為 3:1:0.2 時，以動態光散射儀測定奈米粒粒徑約 100 nm；Zeta 電位約 10 mV，且抗氧化能力佳，並具有 pH 值敏感性，於仿胃酸環境 (pH=1.2) 奈米粒較穩定，而在仿腸道環境 (pH=7.4) 中崩解時釋放可溶性蛋殼膜蛋白達 50%。細胞實驗結果顯示，包覆可溶性蛋殼膜蛋白之奈米粒濃度為 0.8 mg/mL 以下時具有良好生物相容性。以人類腸上皮細胞 (Caco-2) 與小鼠巨噬細胞 (RAW264.7) 共培養系統評估包覆可溶性蛋殼膜蛋白之奈米粒的免疫調節活性，顯示其對脂多醣誘導 RAW264.7 細胞具有降低 NO 量及抑制 TNF-α 及 IL-6 之表現量，且能有效清除 RAW264.7 細胞中的 ROS 含量。透過測量細胞跨膜電阻及 Fluorescein isothiocyanate-dextran 之通透量測定 Caco-2 細胞之通透性，結果顯示包覆可溶性蛋殼膜蛋白之奈米粒能夠減少脂多醣誘導腸上皮緊密連接通透性的增加。綜合上述，包覆可溶性蛋殼膜蛋白之幾丁聚醣/褐藻多醣奈米粒具有治療由脂多醣引起的腸上皮細胞受損之潛力。

Intestinal epithelial tight junctions function as barriers; however, bacterially derived lipopolysaccharides (LPS) can cause impaired gut barrier function to lead to the risk of inflammatory bowel disease. Eggshell membrane proteins exhibit potential anti-inflammatory activity; however, their absorbility through the intestinal mucosa is limited by hydrophilicity and macro-property. The purpose of this study was to prepare chitosan/fucoidan nanoparticles (CS/F NPs) as an oral delivery vehicle to carry soluble eggshell membrane protein (SEP) for the treatment of intestinal epithelial cell damage. In this study, SEP was successfully extracted and encapsulated by CS/F NPs. When the weight ratio of CS, F and SEP was 3:1:0.2, the NP was 100 nm with 10 mV zeta potential, and exhibited good antioxidant capacity. The NPs had pH sensitivity, they were stable in simulated gastric acid environment (pH=1.2), and released 50% of SEP when disintegrated in imitating intestinal environment (pH=7.4). In vitro experiments showed that the SEP-loaded CS/F NPs had good biocompatibility when the concentration was lower than 0.8 mg/mL. Using co-culture system of human intestinal epithelial cells (Caco-2) and mouse macrophages cells (RAW264.7) to assess the immunomodulatory activity, the SEP-loaded CS/F NPs effectively reduced the amount of NO and inhibited the performance of TNF-α and IL-6. Moreover, the results of transepithelial electrical resistance measurement showed that SEP-loaded NPs could reduce the intestinal epithelial tight junction permeabilization caused by LPS. Briefly, the SEP-loaded CS/F NPs have the potential to treat the LPS-induced defective intestinal epithelial cells.

摘要 I
Abstract II
目錄 III
圖目錄 IV
表目錄 VI
第一章、前言 1
第二章、文獻回顧 2
2.1 發炎性腸道疾病 2
2.2 腸道疾病與發炎反應 2
2.3 發炎性腸道疾病治療 5
2.4 蛋殼膜 6
2.5 幾丁聚醣/褐藻多醣奈米粒 7
第三章、實驗目的 10
第四章、實驗架構 11
第五章、實驗流程 12
第六章、實驗材料與方法 13
6.1 實驗材料 13
6.2 實驗藥品 13
6.3 實驗儀器 15
6.4 實驗溶液配製 18
6.5 實驗方法 21
第七章、結果與討論 34
7.1 蛋殼膜成分分析 34
7.2 可溶性蛋殼膜蛋白的製備 34
7.3 幾丁聚醣/褐藻多醣奈米粒之物理化學特性 36
7.4 包覆可溶性蛋殼膜蛋白之奈米粒物化特性 40
7.5 仿腸胃道 pH 值下控制釋放 49
7.6 奈米粒的抗氧化能力測試 50
7.7 細胞實驗 53
第八章、結論 63
第九章、參考文獻 64
第十章、附錄 74

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