臺灣博碩士論文加值系統

目錄
摘要 I
Abstract II
目錄 III
圖目錄 IX
附錄目錄 XI
前言 1
壹、文獻回顧 2
一、皮膚構造及功能 2
1. 表皮 (Epidermis) 2
1-1 角質層 (Stratum corneum) 2
1-2 透明層 (Stratum lucidum) 2
1-3 顆粒層 (Stratum granulosum) 3
1-4 棘狀層 (Stratum spinosum) 3
1-5 基底層 (Stratum basale) 3
2. 真皮層 (Dermis) 3
2-1 乳頭層 (Papillary layer) 4
2-2 網狀層 (Reticular layer) 4
3. 皮下組織 (Subcutaneous tissue) 4
二、異位性皮膚炎 (Atopic dermatitis) 4
1. 異位性皮膚炎介紹 4
2. 異位性皮膚炎之病因及機制 5
3. 異位性皮膚炎之診斷方式 6
4. 異位性皮膚炎之治療藥物 6
4-1 皮質類固醇 (Corticosteroids) 6
4-2 免疫抑制劑 (Immunosuppressant) 7
4-3 抗組織胺藥物 (Anti-histamine) 7
三、肥大細胞脫顆粒作用 (Degranulation) 7
四、化合物 48/80 (Compound 48/80) 8
五、誘導異位性皮膚炎之動物模式 8
1. 經表皮致敏誘發異位性皮膚炎模型 8
1-1 卵白蛋白 (Ovalbumin, OVA) 8
1-2 半抗原 (Hapten) 9
1-3 居家塵蟎 (House dust mite, HDM) 過敏原 9
1-4 食品過敏小鼠模型 9
1-5 超級抗原 (Superantigen) 10
1-6 維生素 D 及其類似物 10
2. 自發性異位性皮膚炎小鼠模型 10
3. 基因工程小鼠異位性皮膚炎模型 11
六、腸道菌相 11
七、海藻 12
1. 海藻簡介 12
2. 綠藻植物門 (Chlorophyta) 12
2-1 石蓴 (Ulva lactuca) 13
3. 海藻多醣 14
3-1 石蓴多醣生理活性 14
貳、實驗設計流程 16
一、石蓴萃取與特性分析 16
二、細胞實驗 16
三、動物實驗 17
參、實驗材料與方法 19
一、實驗材料 19
1. 樣品原料及萃取用溶液 19
2. 樣品成分分析試驗 19
3. 抗氧化試驗 19
4. 細胞株 20
5. 細胞培養之培養基 20
6. 細胞存活率試劑 20
7. P815 脫顆粒測定 20
8. 一氧化氮試驗 20
9. 實驗動物 21
10. 動物實驗誘導 21
11. 組織切片 21
12. 酵素連結免疫吸附分析法 21
13. RNA 萃取套組 21
14. 反轉錄反應試劑 22
15. 即時定量聚合酶鏈鎖反應試劑 (Real-time PCR) 22
16. 糞便 DNA 萃取套組 22
二、儀器設備 23
三、實驗方法 24
1. 實驗樣品製備 24
1-1 熱水萃取法 24
2. 樣品特性分析 24
2-1 膠體滲透層析法 (Gel Permeation Chromatography, GPC) 24
2-2 硫酸根含量測定 24
2-3 總醣含量測定 (Total sugar content) 25
2-4 總酚含量測定 (Total phenolic content) 25
2-5 抗氧化能力試驗 25
2-5-1 清除DPPH (α,α-diphenyl-β-picryhydrazyl) 自由基能力測定 25
2-5-2 清除超氧陰離子能力測定 26
2-5-3 螯合亞鐵離子能力測定 26
3. 細胞實驗 (In vitro) 26
3-1 細胞培養 (Cell culture) 26
3-2 細胞保存 27
3-3 細胞活化 27
3-4 肥大細胞毒性測試 (Cytotoxicity test) 27
3-5 肥大細胞脫顆粒測定 (Mast cell degranulation test) 27
3-6 巨噬細胞毒性測試及細胞內一氧化氮 (Nitrate oxide, NO) 含量測定 28
3-7 酵素連結免疫吸附分析法 (Enzyme-linked immunoadsorbent assay, ELISA) 28
3-8 分析細胞中細胞激素之mRNA 表現量 28
3-8-1 抽取細胞 total RNA 29
3-8-2 反轉錄反應 (Reverse transcription, RT) 29
3-8-3 即時聚合酶連鎖反應 (Real-time polymerase chain reaction) 29
3-9 P815 細胞與 RAW 264.7 細胞共培養 30
4. 動物實驗 (In vivo) 30
4-1 誘導異位性皮膚炎小鼠動物模式 30
4-2 口服石蓴多醣減緩異位性皮膚炎小鼠之效果 31
4-3 組織病理石蠟切片 31
4-3-1 蘇木素-伊紅染色 (Hematoxylin and eosin stain, H&E stain) 31
4-3-2 吉姆薩染色 (Giemsa stain) 32
4-4 血液分析 32
4-5 酵素連結免疫吸附分析法 (Enzyme-linked immunoadsorbent assay, ELISA) 32
4-6 分析組織中細胞激素之mRNA 表現量 32
4-6-1 抽取組織total RNA 32
4-6-2 反轉錄反應 (Reverse transcription, RT) 33
4-6-3 即時聚合酶連鎖反應 (Real-time polymerase chain reaction) 33
4-7 腸道菌相分析 34
5. 統計分析 34
肆、結果 35
一、石蓴水萃物成分與生物活性分析 35
1. 石蓴水萃物之成分分析 35
2. 石蓴水萃物之抗氧化能力分析 35
二、石蓴水萃物對於體外實驗之影響 35
1. 石蓴水萃物對於 P815 老鼠肥大細胞之效果 36
1-1 細胞毒性測試 (Cytotoxicity test) 36
1-2 石蓴水萃物對 Compound 48/80 誘導之肥大細胞脫顆粒之效果測定 36
1-3 石蓴水萃物對 Compound 48/80 誘導之肥大細胞產生細胞激素之影響 36
2. 石蓴水萃物對於 RAW 264.7 老鼠巨噬細胞之效果 36
2-1 細胞毒性測試 (Cytotoxicity test) 37
2-2 一氧化氮釋放試驗 37
3. 石蓴水萃物對於 P815/RAW 264.7 共培養模型之影響 37
3-1 含石蓴水萃物之P815 誘導後上清液對於 RAW 264.7 巨噬細胞之影響 37
3-2 含石蓴水萃物之RAW 264.7 誘導後上清液對於 P815 肥大細胞之影響 38
3-3 石蓴水萃物對於 RAW 264.7 細胞激素之基因表達影響 38
三、石蓴水萃物對於體內實驗之影響 39
1. 皮膚損傷評分及淋巴結重量 39
2. 皮膚組織切片 39
3. 酵素連結免疫吸附分析法 (Enzyme-linked immunoadsorbent assay, ELISA) 40
4. 皮膚細胞激素 41
5. 石蓴水萃物對於腸道菌相之改善效果 41
伍、討論 42
一、石蓴水萃物成分與生物活性分析 42
二、以體外試驗探討石蓴水萃物之生理活性 43
1. 探討石蓴水萃物對於 P815 老鼠肥大細胞之影響 43
2. 石蓴水萃物對於 RAW 264.7 老鼠巨噬細胞之影響 44
3. 石蓴水萃物對於RAW 264.7 老鼠巨噬細胞與P815 老鼠肥大細胞共培養實驗之影響 44
三、以體內試驗探討石蓴水萃物之生理活性 46
陸、結論 50
柒、參考文獻 51
捌、圖 63
玖、附錄 84

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