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詳目顯示區塊

第 1 筆
| (以作者名查詢海洋大學圖書館館藏系統)
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| (以作者名查詢全國博碩士系統)
論文名稱: | 探討岩藻寡醣之最適生產條件與生理活性與 經人類腸癌細胞株 Caco-2 吸收及運輸之變化
| Study on the Optimal Production Conditions and Bioactivities of Fucooligosaccharides and It’s Performance on Absorption and Transportation with Human Colon Adenocarcinoma Cell Caco-2
研究生: | 張劭強
指導教授: | 潘崇良 Chorng-Liang Pan
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | 10032060
畢業學年度: | 101
語文別: | 中文
關鍵字: | 岩藻寡醣
| 生理活性
| MDA-MB-231 細胞
| Caco-2 細胞
| 腸道細胞吸收運輸
全文影像連結: | | (本論文20160728開放校外瀏覽)
| 論文全文(pdf)

論文頁數: | 121
摘要: | 本研究目的為探討岩藻寡醣之最適生產條件與相關生理活性之影響，以及於人類結腸癌細胞株 Caco-2 建立之腸道模式下，評估岩藻寡醣於腸道吸收及運輸之變化。自莢托馬尾藻萃取岩藻聚醣，比較程序 a (先以 Fucosidase rfuc I-H 水解 4 hr，失活再加入 MA103 與 MAEF108 粗酵素液水解 24 hr) 與程序 b (同時添加 Fucosidase rfuc I-H、MA103 與 MAEF108 粗酵素液水解 24 hr) 水解岩藻聚醣所得岩藻寡醣 Fuc-S1 與 Fuc-S2 含量，Fuc-S1 含量於 2 程序下分別為 12.6% 與 13.1%，Fuc-S2 含量則分別為 86.2% 與 85.9%；二者程序水解所得寡醣含量相近，經換算 Fuc-S1 與 Fuc-S2 產率為每 100 g 海藻乾重中含 0.70 g 與 4.65 g 岩藻寡醣。由於程序 a 水解較為耗時，因此將以程序 b 做為岩藻寡醣最適生產條件。將酵素水解岩藻聚醣所得之水解液利用超過濾系統區分出小於 3 kDa 分子量之寡醣水解液，經過 Sephadex G-10 膠體過濾層析 (Gel permeation chromartography, GPC) 可獲得岩藻寡醣 Fuc-S1 與 Fuc-S2。分別收集區分液凍乾，利用高效能液相層析儀 (High performance liquid chromatograph, HPLC) 分析，初步推估 Fuc-S1 為聚合度 DP3，而 Fuc-S2 為 DP2。測定 Fuc-S1 與 Fuc-S2 之硫酸基含量，分別為 0.28 ± 0.01%、0.49 ± 0.01%。利用傅立葉轉換紅外線光譜儀 (Fourier transform infraRed spectroscopy, FT-IR) 分析岩藻寡醣 Fuc-S1 與 Fuc-S2 結構官能基，波長數 3,440 cm-1 有 O-H 官能基，以及在波長數 2,870 cm-1 之 C-H 官能基，另外於波長數 1,248-1,251 cm-1 (S=O) 與 823-829 cm-1 (C-O-S) 之硫酸基團訊號。進一步以 NMR 分析岩藻寡醣 Fuc-S1 與 Fuc-S2 之結構，二者顯示分別在化學位移 1.22 ppm 與 1.28 ppm 之 a-L-Fucp (H6) 之信號，此訊號也確認岩藻寡醣 Fuc-S1 與 Fuc-S2 為 (1→3) 鍵結之岩藻寡醣，而 H1 信號在化學位移 4.33 ppm 與 4.67 ppm，H2-H5 信號在化學位移集中在 3.2-4.0 ppm。抗凝血試驗中之 APTT 於兔血漿及人血漿部分，於濃度 5 mg/mL 與 20 mg/mL 之岩藻寡醣 Fuc-S1 與 Fuc-S2，可較空白組延遲約 22-77 sec 與 11-22 sec，且可降低凝血程度。在 TT 試驗，同樣濃度之 Fuc-S1 與 Fuc-S2 於兔血漿部分可較空白組延遲 11sec 與 22-44 sec。岩藻寡醣 Fuc-S1 與 Fuc-S2 於抗血小板凝集試驗上，濃度 5 mg/mL 與 20 mg/mL 之 Fuc-S1 可較自願者 A (控制組) 分別降低 1.5% 與 3.4%，較自願者 B 降低 0.3% 與 1.0% 之血小板凝集百分比；Fuc-S2 於相同作用濃度下則可較自願者 A 降低 1.4% 與 8.1%，較自願者 B 降低 5.3% 與 9.1% 之血小板凝集百分比。於抗氧化試驗，岩藻寡醣 Fuc-S1 與 Fuc-S2 分別有 26.47 ± 0.01% 與 9.78 ± 0.01% 清除 DPPH 自由基能力，相當於 31.42 ± 0.02 與 9.84 ± 0.01 ug/mL Trolox。在螯合亞鐵離子能力方面，Fuc-S1 具有 32.53 ± 0.04% 之 螯合能力。在評估抑制人類乳癌細胞 MDA-MB-231 遷移效果之傷口癒合試驗結果顯示，岩藻寡醣 Fuc-S1 與 Fuc-S2 在反應濃度為 0.8 mg/mL 時之 MDA-MB-231 細胞遷移距離分別為 460 m 與 462 m；二者在反應濃度 8 mg/mL 時，岩藻寡醣 Fuc-S1 與 Fuc-S2 之細胞遷移距離分別為 235 m 與 300 m，與控制組 (715 m) 相比呈現的抑制 MDA-MB-231 細胞遷移之效果。評估岩藻寡醣 Fuc-S1 與 Fuc-S2 抑制登革熱病毒第 2 型對宿主細胞吸附反應之影響，經岩藻寡醣 Fuc-S1 以濃度 100 ug/mL 與 1 ug/mL 反應之 BHK-21 細胞存活率分別為 80% 與 78%，而同樣濃度下，岩藻寡醣 Fuc-S2 反應之細胞存活率分別為 86% 與 83%，與控制組 (存活率 74%) 相比具抑制病毒吸附之效果。岩藻寡醣 Fuc-S1 與 Fuc-S2 經 Caco-2 細胞吸收運輸，經 HPLC 分析岩藻寡醣 Fuc-S1 之上層液中醣含量從 3.71 ± 0.73 mg/mL 降至 3.04 ± 0.34 mg/mL，下層液中含量則從 2.33 ± 0.31 mg/mL 上升至 2.80 ± 0.53 mg/mL，而被 Caco-2 細胞吸收之含量相似約在 4.07 ± 0.60 mg/mL；岩藻寡醣 Fuc-S2 之上層液岩藻寡醣含量自 2.93 ± 0.24 mg/mL 至 2.14 ± 0.86 mg/mL，下層液則從 2.15 ± 0.18 mg/mL 至 3.10 ± 0.27 mg/mL，而被 Caoc-2 細胞吸收之含量約 4.48 ± 0.80 mg/mL。以反應 90 min 之結果可知，岩藻寡醣 Fuc-S1 其運輸率推估約為 28.1%，Caco-2 細胞吸收率約為 39.0 %；而 Fuc-S2 其運輸率約為 31%，Caco-2 細胞吸收率約為 47.6%。
論文目次: | 中文摘要    I
| 英文摘要 (Abstract)    III
| 目錄    V
| 圖目錄    IX
| 表目錄    XI
| 附錄目錄    XII
| 壹、前言    1
| 貳、文獻整理    3
| 一、海藻簡介    3
| 二、海藻多醣    3
| 2-1. 海藻多醣定義    3
| 2-2. 海藻多醣之生理活性    4
| 2-2-1. 抗凝血    4
| 2-2-2. 抗氧化    4
| 2-2-3. 抗病毒    4
| 2-2-4. 抗腫瘤    5
| 三、海藻寡醣    5
| 3-1. 海藻寡醣之萃取方法    5
| 3-2. 海藻寡醣之生理活性    6
| 3-2-1. 抗氧化    6
| 3-2-2. 抗凝血    6
| 3-2-3. 抗病毒    7
| 3-2-4. 抗腫瘤    7
| 四、岩藻聚醣 (Fucoidan)    7
| 4-1. 岩藻聚醣的組成與結構    7
| 4-2. 岩藻聚醣之萃取方法    8
| 4-2-1. 酸萃取法    8
| 4-2-2. 水萃取法    8
| 4-2-3.  CaCl2 溶液萃取    8
| 4-3. 岩藻聚醣及岩藻寡醣之生理活性    9
| 4-3-1. 抗發炎    9
| 4-3-2. 抗腫瘤    9
| 4-3-3. 抗凝血    9
| 4-3-4. 抗氧化    9
| 4-3-5. 抗病毒    10
| 五、岩藻聚醣水解酵素    10
| 六、腸道細胞株 Caco-2    10
| 6-1. Caco-2 細胞簡介    10
| 6-2. Caco-2 細胞之吸收模式    11
| 6-2-1. 藥物吸收    11
| 参、實驗設計    12
| 肆、材料方法    13
| 一、實驗材料    13
| 1-1. 原料    13
| 1-2. 實驗菌株    13
| 1-3. 實驗細胞株    13
| 1-4. 實驗病毒株    13
| 1-5. 試驗藥品    13
| 1-5-1. 藥品    13
| 1-5-3. 菌株培養基組成    15
| 1-5-4. 細胞培養液組成    16
| 1-5-5.    Fucosidase 反應基質    16
| 1-5-6. 無菌過濾    17
| 1-5-7. 薄層層析矽膠片    17
| 1-6. 儀器設備    17
| 二、實驗方法    18
| 2-1. 菌株保存與活化    18
| 2-1-1. 菌株保存    18
| 2-1-2. 菌株活化    18
| 2-2. 莢托馬尾藻之一般成份分析    18
| 2-2-1. 水分含量    19
| 2-2-2. 灰分含量    19
| 2-2-3. 粗蛋白含量    19
| 2-2-4. 粗脂肪含量    19
| 2-3. 岩藻聚醣之萃取與 Fucosidases 製備    19
| 2-3-1. 岩藻聚醣之酸萃取    19
| 2-3-2. 以岩藻聚醣誘導菌株 MA103、MAEF108 生產 Fucosidases    粗酵素液    20
| 2-3-3. 以 Nisin 誘導Lc. lactis rfucI-H 生產 Fucosidase rfucI-H    20
| 2-3-4.      Fucosidase 酵素活性分析    20
| 2-4. 岩藻寡醣之分離與純化    20
| 2-4-1. 不同水解處理程序水解岩藻聚醣    20
| 2-4-2. 超過濾 (Molecular weight cut off, MWCO)    21
| 2-4-3. 膠體過濾層析 (GPC)    21
| 2-5. 岩藻寡醣之成分分析    21
| 2-5-1. 總醣量測定    21
| 2-5-2. 還原醣量測定    21
| 2-5-3. 硫酸酯含量測定    21
| 2-5-4. 總酚含量測定    22
| 2-5-5. 薄層層析 (TLC)    22
| 2-5-6. 高效能液相層析 (HPLC)    22
| 2-5-7. 傅立葉轉換紅外線光譜 (FT-IR) 分析    23
| 2-5-8. 核磁共振光譜儀 (NMR) 分析    23
| 2-6. 抗氧化活性測定    23
| 2-6-1. 清除 2,2- Diphenyl-1-Pikryl-hydrazyl (DPPH) 自由基能力    之測定    23
| 2-6-2. 還原能力之測定    23
| 2-6-3. 亞鐵離子螯合能力之測定    23
| 2-7. 抗凝血活性測定    24
| 2-7-1. 活化部份凝血活酶時間 (Activited partial thromboplastin time, APTT)    24
| 2-7-2. 凝血酶原時間 (Prothrombin time, PT)    24
| 2-7-3.  凝血酶時間 (Thromboplastin time, TT)    24
| 2-7-4. 富含血小板血漿 (Platelet-rich plasma, PRP) 之製備    24
| 2-7-5. 血小板凝集測定 (Platelet aggregation)    25
| 2-8. 評估岩藻寡醣抗病毒能力    25
| 2-8-1. 細胞株培養方法及繼代方法    25
| 2-8-2. 細胞之冷凍保存    25
| 2-8-3. 細胞毒性試驗 (MTT assay)    25
| 2-8-4. 抗登革熱病毒試驗    26
| 2-8-4-1抑制病毒對宿主細胞吸附反應    26
| 2-9. 岩藻寡醣抑制人類乳癌細胞 MDA-MB-231 遷移能力    26
| 2-9-1. 細胞株培養方法及繼代方法    26
| 2-9-2. 細胞之冷凍保存    27
| 2-9-3. 傷口癒合試驗 (Wound healing assay)    27
| 2-10. 岩藻寡醣於腸道細胞吸收與運輸試驗    27
| 2-10-1.  Caco-2 細胞株之解凍    27
| 2-10-2.  Caco-2 細胞株培養方法及繼代方法    27
| 2-10-3. 細胞之冷凍保存    28
| 2-10-4.  Caco-2 細胞存活率分析 (MTT assay)    28
| 2-10-5.  Transwell insert 培養法    28
| 2-10-6.  Caco-2 細胞單層膜完整性測試    28
| 2-10-7. 岩藻寡醣 Fuc-S1 與 Fuc-S2 於腸道細胞吸收運輸試驗    29
| 2-11. 岩藻寡醣經腸道吸收及運輸分析    29
| 2-11-1. 測定含有岩藻寡醣運輸液之總醣量    29
| 2-11-2. 含有岩藻寡醣運輸液之 HPLC 分析    29
| 2-11-3. 測定 Caco-2 細胞吸收之總醣量    29
| 2-11-4. 經 Caco-2 細胞吸收之岩藻寡醣 HPLC 分析    29
| 2-12. 統計分析    30
| 伍、結果與討論    31
| 陸、結論    42
| 柒、參考文獻    44
|
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2013 年 8 月 5 日 22:54