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| | | | | 第 | | 筆

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)
| (以作者名查詢全國圖書書目資訊網)
| (以作者名查詢全國博碩士系統)
論文名稱: | 探討馬尾藻與螺旋藻寡醣混合物對PC12細胞因砷誘發氧化壓力之影響
| Study on the Influence of Algae Oligosaccharides Derived from Sargassum and Spirulina on Arsenic–induced Oxidative Stress in PC12 Cell Lines
研究生: | 范素芬 Su-Fen Fan
指導教授: | 潘崇良 Chorng-Liang Pan
| 林滿玉 Anya Maan-Yuh Lin
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: | T96420006
畢業學年度: | 97
語文別: | 中文
關鍵字: | 馬尾藻 Spirulina
| 螺旋藻 Sargassum
| PC12 PC12
| 砷 Arsenic
| 氧化壓力 Oxidative stress
全文影像連結: | | (本論文20120730對校內公開)
| 論文全文(pdf)

論文頁數: | 103
摘要: | 摘  要
|
| 本研究以Pseudomonas vesicularis MA103及Aeromonas salmonicida MAEF108之agarases 粗酵素液，水解馬尾藻與螺旋藻，取得＜ 1 kDa、1-3 kDa、與 3-5 kDa 馬尾藻與螺旋藻寡醣混合物，進行抗氧化活性測試，並探討馬尾藻與螺旋藻寡醣混合物可否降低砷對 PC12 神經細胞株造成的氧化壓力。結果顯示 3-5 kDa 的馬尾藻寡醣混合物在總醣量及還原醣量皆為最高，分別為 870.94 ± 67.16 g/mL及0.62 ± 0.01 mg/mL；硫酸基含量 (%)則以 3-5 kDa 螺旋藻寡醣混合物較高 (32.20 ± 0.11%)。抗氧化能力方面，總酚含量以 3-5 kDa 的馬尾藻寡醣混合物含量較高 (86.67 ± 0.88 g/mL)；在清除 DPPH 自由基方面以馬尾藻寡醣混合物表現較佳，介於 22.60 ± 2.10% 至 11.34 ± 1.98%之間；但亞鐵離子螯合效應以＜ 1 kDa 螺旋藻寡醣混合物 (81.61 ± 0.13%)表現較佳。在細胞實驗方面，1-3 kDa 的馬尾藻寡醣混合物或＜ 1 kDa、1-3 kDa、及 3-5 kDa 的螺旋藻寡醣混合物 (0.032 mg/mL)，可以明顯降低 50 M As3+ 造成 PC12 細胞死亡的情形，與As3+組比較分別增加 7.2%、11.9%、7.4%、及13.6%細胞存活率。進一步發現PC12細胞經50 M As3+處理24 hr，會明顯增加細胞內活性氧的含量，但同時給予 1-3 kDa 的馬尾藻寡醣混合物或＜ 1 kDa、1-3 kDa、及3-5 kDa 的螺旋藻寡醣混合物 (0.032 mg/mL)，可以抑制活性氧的生成，與As3+比較分別可降低 55%、61%、58%、及52%活性氧含量。接著探討 PC12細胞經 As3+ 處理後，細胞內麩胱甘肽含量的變化，結果發現：PC12細胞經As3+處理且同時給予 1-3 kDa 馬尾藻寡醣混合物、＜ 1 kDa、1-3 kDa、或 3-5 kDa 螺旋藻寡醣混合物 (0.032 mg/mL)，可以增加細胞內麩胱甘肽的含量，與As3+比較分別增加 98%、89%、87%、及 115%。另外以西方墨點法分析熱休克蛋白 70 (Heat shock protein 70, HSP 70)與血紅素加氧酵素-1 (Heme oxygenase-1, HO-1)的表現，發現1-3 kDa 馬尾藻寡醣混合物 (0.032 mg/mL)可以抑制 As3+ 所誘導ROS，進而減少 HSP 70 與 HO-1的表現量，與 As3+ 比較明顯降低13% 及 14%；As3+ 與 3-5 kDa 螺旋藻寡醣混合物組 HSP 70 和HO-1 表現量，與 As3+ 組比較明顯降低 50% 及 48%。PC12 細胞經 50 M As3+ 處理 24 hr，磷酸化 Extracellular signal-regulated kinases (ERK)會明顯增加，但無論給予馬尾藻或螺旋藻寡醣混合物 (0.032 mg/mL)，都無法明顯降低磷酸化 ERK 的表現；在磷酸化 c-Jun N-terminal kinases (JNK)的變化方面，給予 As3+ 和 1-3 kDa 馬尾藻、1-3 kDa、或 3-5 kDa 螺旋藻寡醣混合物 (0.032 mg/mL)，與As3+比較可以明顯降低磷酸化 JNK 的表現量 (33%至46%)；最後 PC12細胞經 50 M As3+ 和 1-3 kDa 馬尾藻、＜ 1 kDa、1-3 kDa、或 3-5 kDa 螺旋藻寡醣混合物 (0.032 mg/mL)處理 24 hr 後，磷酸化 P38 可以明顯降低 (28%至39%)。由以上結果得知：馬尾藻與螺旋藻寡醣混合物可以降低As3+在PC12細胞內誘發的氧化壓力，減少PC12細胞死亡，未來具有開發成抗氧化劑之潛力，做為一種新型態保健食品開發之基礎。
|
| Abstract
|
| In the present study, Sargassum and Spirulina polysaccharides extracts were degraded by Pseudomonas vesicularis MA103 and Aeromonas salmonicida MAEF108 agarases to yield Sargassum and Spirulina oligosaccharides hydrolysate fraction of < 1 kDa, 1-3 kDa, and 3-5 kDa to perform anti-oxidative activity assays. And the better anti-oxidative algal samples are following evaluated whether treatment with Sargassum or Spirulina oligosaccharides hydrolysate fraction reduced arsenic-induced oxidative stress in PC12 neuronal cells. Experimental data showed that 3-5 kDa Sargassum oligosaccharides hydrolysate fraction contained the highest total sugar content and reducing sugar content (870.94 ± 67.16  g/mL and 0.62 ± 0.01 mg/mL), and 3-5 kDa Spirulina oligosaccharides hydrolysate fraction contained the highest sulfated content (32.20 ± 0.11%). In terms of anti-oxidative activities, the total phenolic content was highest in the 3-5 kDa Sargassum oligosaccharides hydrolysate fraction (86.67 ± 0.88 g/mL). In evaluating the scavenging effects on DPPH, Sargassum oligosaccharides hydrolysate fraction performed better, while < 1 kDa Spirulina oligo- saccharides hydrolysate fraction had better chelating effects on ferrous ions. In vitro cell studies showed that As3+-treated PC12 neuronal cells had significantly reduced cell viability, while those cotreated with 1-3 kDa Sargassum, < 1 kDa, 1-3 kDa, or 3-5 kDa Spirulina oligosaccharides hydrolysate fraction had significantly increased cell viability relative to As3+ only group, with increases of 7.2%, 11.9%, 7.4%, and 13.6% cell viability, respectively. Additionally, when given 1-3 kDa Sargassum, <  1 kDa, 1-3 kDa, or 3-5 kDa Spirulina oligosaccharides hydrolysate fraction with As3+, reactive oxygen species (ROS) production of PC12 neuronal cells was inhibited, with decreases of 55%, 61%, 58%, and 52%, respectively, relative to As3+ group. Furthermone, intracellular glutathione (GSH) level changed after treating PC12 neuronal cells with As3+ and results showed that when 1-3 kDa Sargassum, < 1 kDa, 1-3 kDa, or 3-5 kDa Spirulina oligosaccharides hydrolysate fraction was given with As3+, intracellular GSH content of PC12 neuronal cells increased by 98%, 89%, 87%, and 115%, respectively, relative to As3+ group.  Using western blotting to analyze heat shock protein 70 (HSP 70) and heme oxygenase-1 (HO-1) expression, results indicated that 1-3 kDa Sargassum oligosaccharides hydrolysate fraction significantly inhibited As3+-induced HSP 70 and HO-1 expression by 13% and 14% when compared to As3+ group, and 3-5 kDa oligosaccharides hydrolysate fraction inhibited by 50% and 48%.  After treatment with 50 M As3+ for 24 hr, intracellular phosphorylated extracellular signal-regulated kinases (ERK) expression of PC12 neuronal cells significantly increased.  However, regardless of whether Sargassum or Spirulina oligosaccharides hydrolysate fraction was added, phosphorylated ERK expression was not significantly reduced.  In terms of changes in phosphorylated c-Jun N-terminal kinases  (JNK) expression, giving As3+ with 1-3 kDa Sargassum, 1-3 kDa, or 3-5 kDa Spirulina oligosaccharides hydrolysate fraction significantly decreased phosphorylated JNK expression (from 33% to 46%).  Lastly, after treatment of 50 M As3+ with 1-3 kDa Sargassum, < 1 kDa, 1-3 kDa, or 3-5 kDa Spirulina oligosaccharides hydrolysate fraction for 24 hr, phosphorylated P38 expression was noticeably reduced in PC12 neuronal cells (from 28% to 39%). In conclusion, Sargassum and Spirulina oligosaccharides hydrolysate fraction reduce As3+-induced oxidative stress and cell death in PC12 neuronal cells, serving as the basis for development of anti-oxidative agents in the future, thus creating a novel form of health food products.
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論文目次: | 目 錄
| 目錄 ...........................................................................................................i
| 表目錄 ..........................................................................................................v
| 圖目錄 .........................................................................................................vi
| 附錄目錄......................................................................................................viii
| 中文摘要.........................................................................................................ix
| 英文摘要.........................................................................................................xi
| 壹、前言..........................................................................................................................1
| 貳、文獻整理.................................................................................................................3
| 01. 砷化物 .........................................................................................3
| 1-1. 砷化物的來源和分佈........................................................................3
| 1-2. 砷化物的種類與攝取代謝..............................................................3
| 02. 砷影響相關基因之表現....................................................................4
| 2-1. 砷與HO-1關係之探討.....................................................................4
| 2-2. 砷與HSP 70關係之探討.................................................................5
| 2-3. 砷化物對細胞內MAPKs之影響....................................................5
| 03. 抗氧化能力測定...............................................................................6
| 3-1. 自由基與活性氧之形成...................................................................6
| 3-2. 氧化傷害...............................................................................................7
| 3-3. 抗氧化物的種類.................................................................................7
| 04. 腎上腺髓質嗜鉻細胞瘤....................................................................9
| 05. 海藻之來源及應用.........................................................................10
| 5-1. 馬尾藻....................................................................................10
| 5-2. 螺旋藻....................................................................................10
| 06. 洋菜糖 (Agarose) 的構造與特性..................................................11
| 07. 洋菜酶對洋菜水解的作用機制......................................................11
| 08. 海藻寡醣組成成分之鑑定..............................................................12
| ii
| 8-1. 薄層色層層析法 (Thin-layer chromatography, TLC)...........12
| 8-2. 高效能液相層析法 (High-performance liquid chromato- graphy, HPLC)......................................................................................12
| 8-3. 磁核共振光譜法 (Nuclear magnetic resonance, NMR)........13
| 09. 藻類寡醣生理活性..........................................................................14
| 9-1. 免疫性..................................................................................................14
| 9-2. 降低血脂.............................................................................................15
| 9-3. 益菌性..................................................................................................15
| 参、實驗設計...............................................................................................................16
| 肆、實驗材料...............................................................................................................17
| 01. 實驗菌株 ........................................................................................17
| 1-1. 產洋菜酶海洋菌株..........................................................................17
| 1-2. 產洋菜酶海洋菌株之保存............................................................17
| 1-3. 產洋菜酶海洋菌株之活化............................................................17
| 02. 洋菜酶反應基質..............................................................................17
| 03. 實驗試劑 ........................................................................................18
| 04. 西方墨點法抗體.............................................................................20
| 4-1. 一級抗體.............................................................................................20
| 4-2. 二級抗體.............................................................................................21
| 05. 儀器設備 ........................................................................................21
| 伍、實驗方法...............................................................................................................23
| 01. 洋菜酶之生產.................................................................................23
| 02. 洋菜酶之濃縮.................................................................................24
| 03. 洋菜酶活性測定.............................................................................24
| 04. 海藻寡醣水解液之生產.................................................................24
| 05. 藻類之ㄧ般成分分析......................................................................25
| 5-1. 總醣量.................................................................................................25
| iii
| 5-2. 還原醣量之測定...............................................................................25
| 5-3. 硫酸基含量之測定..........................................................................25
| 5-4. 總酚含量.............................................................................................26
| 06. 藻類水解液的生理活性..................................................................26
| 6-1. 螯合亞鐵離子測定..........................................................................26
| 6-2. 清除DPPH自由基能力測定..........................................................27
| 07. 抗凝血活性.....................................................................................27
| 7-1. 凝血酶原時間....................................................................................27
| 7-2. 活化部份凝血活酶時間.................................................................27
| 08. 細胞實驗 ........................................................................................28
| 8-1. PC12細胞培養.................................................................................28
| 8-2. 活化冷凍細胞....................................................................................29
| 8-3. 冷凍保存細胞....................................................................................29
| 09. 細胞存活率分析..............................................................................29
| 10. 細胞內ROS測試.............................................................................30
| 11. 麩胱甘肽 (Glutathione, GSH)濃度測定........................................30
| 12. 西方墨點法......................................................................................31
| 12-1. 總蛋白質之萃取............................................................................31
| 12-2. 蛋白質濃度的測定.......................................................................32
| 12-3. SDS-聚丙烯醯胺凝膠電泳........................................................32
| 13. 統計方法 ........................................................................................33
| 陸、結果與討論............................................................................................34
| 01. 冷水萃取馬尾藻與螺旋藻.............................................................34
| 1-1. 總醣量變化........................................................................................34
| 1-2. 抗氧化能力測定...............................................................................34
| 02. 熱水萃取馬尾藻與螺旋藻.............................................................35
| 2-1. 總醣量、還原醣量、與硫酸基含量變化................................35
| iv
| 2-2. 抗氧化能力測定...............................................................................36
| 03. 馬尾藻與螺旋藻寡醣混合物抗凝血活性.....................................37
| 3-1. 活化部分凝血活酶時間.................................................................38
| 3-2. 凝血酶原時間....................................................................................38
| 04. As3+與海藻寡醣混合物對PC12細胞存活率之影響..................38
| 4-1. 馬尾藻..................................................................................................38
| 4-2. 螺旋藻..................................................................................................39
| 05. As3+與海藻寡醣混合物對PC12細胞ROS產生之影響............40
| 06. As3+與海藻寡醣混合物對PC12細胞GSH含量之影響............42
| 07. As3+與海藻寡醣混合物對熱休克蛋白表現量之影響.................43
| 08. As3+與海藻寡醣混合物對MAPKs表現量之影響......................45
| 柒、結論........................................................................................................................48
| 捌、參考文獻...............................................................................................................49
| 玖、附錄........................................................................................................................96
| v
| 表 目 錄
| 0表1、 5% 與 10% (w/v) 馬尾藻冷水萃取液 pH值及總醣量...........62
| 0表2、 2.5% (w/v) 螺旋藻冷水萃取液 pH值及總醣量........................63
| 0表3、 5% 與10% (w/v) 馬尾藻冷水萃取液總酚含量、對 DPPH
| 自由基之清除效應、及對亞鐵離子螯合效應............................64
| 0表4、 2.5% (w/v) 螺旋藻冷水萃取液總酚含量、對 DPPH 自由
| 基之清除效應、及對亞鐵離子螯合效應....................................65
| 0表5、 10% (w/v) 馬尾藻寡醣混合物總醣量、還原醣量、及硫酸
| 基含量............................................................................................66
| 0表6、 2.5% (w/v) 螺旋藻寡醣混合物總醣量、還原醣量、及硫酸
| 基含量.............................................................................................67
| 0表7、 10% (w/v) 馬尾藻寡醣混合物總酚含量、對DPPH自由基
| 之清除效應、及對亞鐵離子螯合效應........................................68
| 0表8、 2.5% (w/v) 螺旋藻寡醣混合物總酚含量、對DPPH自由基
| 之清除效應、及對亞鐵離子螯合效應........................................69
| 0表9、 馬尾藻寡醣混合物對As3+在PC12細胞中減少麩胱甘肽
| 含量之影響.....................................................................................70
| 表10、 螺旋藻寡醣混合物對As3+在PC12細胞中減少麩胱甘肽
| 含量之影響.....................................................................................71
| 表11、 馬尾藻寡醣混合物對As3+在PC12細胞中活化 HSP 70及
| HO-1表現之影響..........................................................................72
| 表12、 螺旋藻寡醣混合物對As3+在PC12細胞中活化 HSP 70及
| HO-1表現之影響..........................................................................73
| 表13、 馬尾藻寡醣混合物對As3+在PC12細胞中活化 MAPKs
| 表現之影響.....................................................................................74
| 表14、 螺旋藻寡醣混合物對As3+在PC12細胞中活化 MAPKs
| 表現之影響.....................................................................................75
| vi
| 圖 目 錄
| 0圖1、 馬尾藻寡醣混合物對活化部分凝血活酶時間之影響................76
| 0圖2、 螺旋藻寡醣混合物對活化部分凝血活酶時間之影響................77
| 0圖3、馬尾藻寡醣混合物對凝血酶原時間之影響................................78
| 0圖4、螺旋藻寡醣混合物對凝血酶原時間之影響................................79
| 圖5、 ＜ 1 kDa馬尾藻寡醣混合物對As3+在PC12細胞中引起
| 細胞死亡之影響............................................................................80
| 圖6、 1-3 kDa馬尾藻寡醣混合物對As3+在PC12細胞中引起細胞
| 死亡之影響....................................................................................81
| 圖7、 3-5 kDa馬尾藻寡醣混合物對As3+在PC12細胞中引起細胞
| 死亡之影響....................................................................................82
| 圖8、 ＜ 1 kDa螺旋藻寡醣混合物對As3+在PC12細胞中引起
| 細胞死亡之影響............................................................................83
| 圖9、 1-3 kDa螺旋藻寡醣混合物對As3+在PC12細胞中引起細胞
| 死亡之影響....................................................................................84
| 圖10、 3-5 kDa螺旋藻寡醣混合物對As3+在PC12細胞中引起細胞
| 死亡之影響....................................................................................85
| 圖11、 馬尾藻寡醣混合物對As3+在PC12細胞中活性氧產生之
| 影響................................................................................................86
| 圖12、 螺旋藻寡醣混合物對As3+在PC12細胞中活性氧產生之
| 影響................................................................................................87
| 圖13、 馬尾藻寡醣混合物對As3+在PC12細胞中活化HSP 70及
| HO-1表現之影響..........................................................................88
| 圖14、 螺旋藻寡醣混合物對As3+在PC12細胞中活化HSP 70及
| HO-1表現之影響..........................................................................89
| 圖15、 馬尾藻寡醣混合物對As3+在PC12細胞中活化ERK表現之
| 影響................................................................................................90
| vii
| 圖16、 馬尾藻寡醣混合物對As3+在PC12細胞中活化JNK表現之
| 影響................................................................................................91
| 圖17、 馬尾藻寡醣混合物對As3+在PC12細胞中活化P38表現之
| 影響................................................................................................92
| 圖18、 螺旋藻寡醣混合物對As3+在PC12細胞中活化ERK表現之
| 影響................................................................................................93
| 圖19、 螺旋藻寡醣混合物對As3+在PC12細胞中活化JNK表現之
| 影響................................................................................................94
| 圖20、 螺旋藻寡醣混合物對As3+在PC12細胞中活化P38表現之
| 影響................................................................................................95
| viii
| 附 錄 目 錄
| 附錄1、 Agarose 的基本構造及洋菜酶作用位置...................................96
| 附錄2、凝固試驗流程圖........................................................................ 97
| 附錄3、 ＜ 1 kDa馬尾藻寡醣混合物對As3+在PC12細胞中引起
| 細胞死亡影響之原始數據........................................................ 98
| 附錄4、 1-3 kDa馬尾藻寡醣混合物對As3+在PC12細胞中引起
| 細胞死亡影響之原始數據........................................................ 99
| 附錄5、 3-5 kDa馬尾藻寡醣混合物對As3+在PC12細胞中引起
| 細胞死亡影響之原始數據..................................................... 100
| 附錄6、 ＜ 1 kDa螺旋藻寡醣混合物對As3+在PC12細胞中引起
| 細胞死亡影響之原始數據..................................................... 101
| 附錄7、 1-3 kDa螺旋藻寡醣混合物對As3+在PC12細胞中引起
| 細胞死亡影響之原始數據..................................................... 102
| 附錄8、 3-5 kDa螺旋藻寡醣混合物對As3+在PC12細胞中引起
| 細胞死亡影響之原始數據..................................................... 103
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2010 年 3 月 8 日 22:45