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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)
| (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: | 銀耳寡醣之生產及保健生理活性探討
| Study on Production and Health Bioactivities of Oligosaccharides Derived from Tremella fuciformis Berk
研究生: | 衛曉菁 Hsiao-Ching Wei
指導教授: | 潘崇良 Chorng-Liang Pan
| 張學逸 Shyue-Yih Chang
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: | T96420011
畢業學年度: | 97
語文別: | 中文
關鍵字: | 銀耳 collagen
| 寡醣 fibroblast
| MMPs
| TGF-beta
| wound healing
全文影像連結: | | (本論文20120731對校內公開)
| 論文全文(pdf)

論文頁數: | 118
摘要: | 本研究欲以 P. vesicularis MA103 與 A. MAEF108 之 agarases 粗酵素液，水解銀耳多醣後分離出 < 1 kDa (OS-TF1)、1-3 kDa (OS-TF1-3)、3-5 kDa (OS-TF3-5)寡醣區分液。並以冷水萃取 16 與 24 hr，共同測定其抗氧化能力；而三種分子量區間亦測試抗凝血能力；並進一步探討銀耳寡醣是否具促進小鼠胚胎纖維母 (NIH-3T3) 細胞增生、傷口癒合、以及細胞外基質合成能力。實驗結果得知在全醣量、還原醣與硫酸根含量以 OS-TF3-5 含量最高；為 0.270 ± 0.001 mg/ mL、0.136 ± 0.02 mg/ mL 與 88.70 ± 2.74 mg/ mL。抗氧化能力方面，總酚含量以 OS-TF3-5 的含量最高 (0.058 ± 0.003 mg/mL)；亞鐵離子螯合能力則以冷萃 16 hr 效果優於其他組別 (87.136 ± 5.131%) ；而冷萃 24 hr 的組別在 DPPH 自由基清除能力則效果最好 (13.887 ± 1.329%)。抗凝血活性的實驗以兔血漿添加寡醣測定活化部分凝血活酶時間 (activated partial thromboplastin time, APTT)，三個分子量區間之銀耳寡醣相較於空白組皆具延遲 APTT 的效果；其中又以 OS-TF3-5 效果最好。在細胞增生實驗方面，以 10% FBS 的培養條件下在1 mg/mL 的濃度培養 48 hr 相較於對照組，三個分子量區間的銀耳寡醣皆有顯著差異；但並未對劑量呈現依賴之效應。另外雖未有顯著差異，但三個分子量區間的銀耳寡醣在細胞增生之情況下 collagen 與 fibronectin 的生成量卻較對照組低。觀察三個分子量區間的銀耳寡醣對以三氯醋酸 (TCA) 傷害纖維母細胞的保護與修復作用；相較於對照組，OS-TF1 於 1 mg/mL 濃度處理同時具保護與修復作用並有顯著的差異。另外以 Wound-healing assay (創傷癒合) 觀察纖維母細胞之遷移數量與癒合時間；由結果得知 OS-TF1 之組別細胞生長密度與細胞遷移之數量明顯高於同時添加 1 mg/mL OS-TF1 與 2 M PD98059，以及 1 mg/mL OS-TF1 與 2uM SB203580 之組別；推測 OS-TF 刺激纖維母細胞增生與移行可能參與 ERK 與 p38 pathway 相關。創傷 24 hr 後纖維母細胞在 TGF-b、MMP-2，以及 MMP-9 的分泌量以添加 1 mg/mL，OS-TF1 最多，推測銀耳寡醣因刺激 TGF-b而有助於傷口復原；而 MMP-2、MMP-9 則是因纖維母細胞增加 TGF-b 之分泌以利創傷修復的同時其產量亦增加幫助基質重整。綜合以上結果得知 OS-TF 也許具有促進傷口復原卻不會產生疤痕沉積的能力；可供日後研發相關產品之參考。
| This study explores the Tremella fuciformis Berk polysaccharides extracts were degraded by MA103 and MAEF108 agarases to yield Tremella Fuciformis Berk oligosaccharide hydrolysate and ultrafiltrated into fractions of < 1 kDa(OS-TF1), 1-3 kDa(OS-TF1-3), and 3-5 kDa (OS-TF3-5). Cold water extraction was conducted for 16 and 24 hr, to perform anti-oxidative activity. The ability of anti-coagulation was measured by three molecular weight fractions. This study further investgated OS-TF has the abilities to enhance cell proliferation, help wounds to heal over, and to synthesize extracellular matrix. From the results of the experiments, it was found that the OS-TF3-5 is the highest total sugar content, reducing sugar content, and sulfated group content, which are 0.270 ± 0.001 mg/mL, 0.136 ± 0.002 mg/mL, and 88.70 ± 2.74 mg/mL, respectively. In the aspect of anti-coagulation, the fraction with the highest total phenolic content is OS-TF3-5 (0.058 ± 0.003 mg/mL). As for the chelating effects on ferrous ions, the best result was observed 16 hr cold water extractions from Tremella fuciformis Berk (87.136 ± 5.131%). The 24 hr cold water extractions had the best performance in the scavenging effect on DPPH free radicals (13.887 ± 1.329%). In the anti-coagulation activity experiments, rabbit plasma additive oligosaccharide was used to measure the activated partial thromboplastin time (APTT). Compared with the control group, OS-TF has the effect of delaying APTT in all three molecular weight fractions, among them, OS-TF3-5 is the one with the best effect. The result of cell proliferation experiment, under the culture condition of 10% FBS, with the concentration of 1 mg/mL, compared with the control group, the 48 hr cultured significantly differs with molecular weight interval. But there was no dependency on dosage appearing. Besides, although there is no significant difference, in the three molecular weight fractions, under then condition of the proliferation of OS-TF, the amounts of collagen and fibronectin produced were lower than the control group. By observing the protection and repair actions by OS-TF of TCA harming fibroblast cells, compared with the control group, significant difference was found with OS-TF1, with concentration of 1mg/mL, and there were both protection and repair actions. In addiction, observing the number of migration fibroblast and the time taken to heal over in wound-healing assay, from the results it was found that the number of migration fibroblast was significantly higher in the group with OS-TF1 than the group with 1 mg/mL OS-TF1 and 2 uM PD98059, and the group with 1 mg/mL OS-TF1 and 2 uM SB203580. It can be presumed the fact of OS-TF stimulating the proliferation and migration of fibroblast cells may involve ERK and p38 pathway. 24 hr after being wounded, fibroblast cells secretion in TGF-b, MMP-2, and MMP-9 reached the highest point with 1 mg/mL OS-TF1. It can be presumed that wounds heal fast because of the stimulation of TGF-b by OS-TF. While MMP-2 and MMP-9 help wounds to heal over because fibroblast increase TGF-b secretions. Meanwhile, the amount produced was also increased to help with the reform of extracellular matrix. Summarizing the results above, it is OS-TF1 may have the ability to help wound to heal over without causing scars. This fact can be used as a reference for the development of related products in the future.
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論文目次: | 目錄
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| 目    錄    i
| 表 目 錄    vi
| 圖 目 錄    vii
| 附錄目錄    ix
| 中文摘要    xi
| 英文摘要 (Abstract)    xiii
| 壹、前言    1
| 貳、文獻整理    4
| 一、 銀耳簡介    4
| 1-1. 銀耳之外型及分類    4
| 1-2. 生長條件    4
| 1-3. 商業栽種    4
| 1-4. 化學成分組成    5
| 1-5. 生理機能    7
| 1-5-1. 預防高血脂膽固醇    7
| 1-5-2. 降血糖    8
| 1-5-3. 抗腫瘤與免疫調節活性    8
| 1-5-4. 抗發炎和創傷痊癒的效力    9
| 1-5-5. 超氧化物歧化酶的活性    10
| 二、 皮膚結構、疤痕組成與創傷癒合機制    10
| 2-1. 皮膚的生理結構    10
| 2-2. 皮膚之功能    13
| 2-3. 傷口癒合過程    15
| 2-4. 影響傷口癒合的因素    17
| 2-5. 傷口癒合之訊息傳遞路徑    17
| 三、 膠原蛋白    18
| 3-1. 膠原蛋白的結構與分布    19
| 四、 纖維結合蛋白 (Fibronectin) 的表現    20
| 五、 轉型生長因子 (Transforming growth factor , TGF-)    21
| 六、 基質金屬蛋白酵素 (Matrix metalloproteinases, MMPs)    23
| 參、實驗設計    24
| 肆、實驗材料與方法
| 一、實驗材料
| 1-1. 銀耳(Tremella fuciformis Berk ) 子實體    25
| 1-2. 實驗菌株    25
| 1-2-1. 菌株之保存    25
| 1-2-2. 洋菜酶 (Agarase) 生產菌株活化    26
| 1-3. 洋菜酶反應基質    26
| 1-4. 培養基組成    26
| 1-5.    3,5-dinitrosalicylic acid (DNS) 溶液    27
| 1-6. 試驗藥品與試劑    27
| 1-7. 細胞培養耗材與試劑    29
| 1-8. 實驗相關套組    30
| 1-9. 西方墨點法之抗體    30
| 1-10.電泳膠片配製    31
| 1-11.儀器設備    31
| 二、實驗方法      32
| 2-1. 洋菜酶粗酵素液之製備    32
| 2-2. 洋菜酶之濃縮    32
| 2-3. 洋菜酶的活性測定    33
| 2-4. 銀耳子實體寡醣製備    33
| 2-4-1. 銀耳多醣熱萃液    33
| 2-4-2. 銀耳寡醣水解液之生產    33
| 2-4-3. 銀耳寡醣之乾燥處理    33
| 2-5. 銀耳之一般成份分析    34
| 2-5-1. 水分含量    34
| 2-5-2. 灰分含量    34
| 2-5-3. 粗蛋白含量    34
| 2-5-4. 粗脂肪含量    34
| 2-5-5. 總醣量    35
| 2-5-6. 還原醣量    35
| 2-5-7. 總酚量    36
| 2-5-8. 硫酸根含量    36
| 2-5-9. 清除, -Diphenyl--picrylhydrazl (DPPH)
| 自由基能力之測定    37
| 2-5-10.亞鐵離子螯合能力之測定    38
| 2-6. 凝血酶原時間    38
| 2-7. 活化部份凝血活酶時間    38
| 2-8. 細胞培養與實驗    39
| 2-8-1. 細胞來源    39
| 2-8-2. 纖維母細胞培養之條件    39
| 2-8-3. 細胞之解凍活化    39
| 2-8-4. 細胞之冷凍保存    39
| 2-8-5. 細胞增生實驗    40
| 2-8-6. 銀耳寡醣對促進膠原蛋白(Collagen) 之合成之
| 作用    41
| 2-8-7. 銀耳寡醣對受到TCA傷害之纖維母細胞的修護
| 與保護作用    41
| 2-8-8. 創傷癒合實驗(Wound healing assay)    42
| 2-8-9. 轉型生長因子(Transforming growth factor-, TGF-)
| 之測定    42
| 2-8-10.基質金屬蛋白酶(Matrix metalloproteinases, MMPs)
| 之測定    43
| 2-9. 西方墨點法(Western blot)    43
| 2-9-1. 蛋白質萃取    43
| 2-9-2. 蛋白質濃度測定    44
| 2-9-3.    SDS-聚丙烯醯胺凝膠電泳    44
| 2-10.統計分析    45
| 伍、結果與討論    46
| 5-1. 銀耳之一般成份分析    46
| 5-2. 冷水萃取銀耳子實體    46
| 5-3. 熱水萃取銀耳子實體    46
| 5-3-1. 總醣量、還原醣及硫酸根含量    46
| 5-3-2. 抗氧化能力之測定    47
| 5-4. 凝血酶原時間 (Prothrombin time, PT)    47
| 5-5. 活化部份凝血活酶時間(Activited partial thromboplastin
| Time, APTT)    48
| 5-6. 不同濃度之銀耳寡醣對3T3細胞株增生率的影響    48
| 5-7. 不同分子量區間之銀耳寡醣促進膠原蛋白(Collagen)
| 合成之作用    50
| 5-8. 不同分子量區間之銀耳寡醣促進纖維結合蛋白
| (Fibronectin) 合成作用    51
| 5-9.    銀耳寡醣對受到 TCA 傷害之纖維母細胞的修護與
| 保護作用    51
| 5-10.    ERK抑制劑與p38抑制劑可有效抑制因銀耳寡醣處                理所導致的纖維母細胞增生    52
| 5-11.    創傷癒合實驗 (Wound healing assay)    53
| 5-12.    創傷後 < 1 kDa銀耳寡醣與 ERK、p38抑制劑對於                纖維結合蛋白 (Fibronectin) 合成之影響    54
| 5-13.    轉型生長因子 (TGF-) 之測定    55
| 5-14. 基質金屬蛋白酶 (MMPs) 之測定    56
| 陸、結論    57
| 柒、參考文獻    58
| 捌、附錄    97
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| 表目錄
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| 表1. 市售銀耳一般成分分析    68
| 表2.    5% (w/v) 銀耳冷水萃取液萃取 16與24 hr下之總酚含量、
| 對DPPH自由清除效應、及對亞鐵離子螯合效應之變化    69
| 表3.    5% (w/v) 銀耳多醣熱萃液經1 unit/ mL MAEF108-crude
| agarases 與 MA103-crude agarases水解 24 hr後之總醣量
| 與還原醣量    70
| 表4.    5% (w/v) 銀耳多醣熱萃液經1 unit/ mL MAEF108-crude
| agarases 與 MA103-crude agarases水解 24 hr後之pH
| 及硫酸根含量    71
| 表5.    5% (w/v) 銀耳多醣熱萃液以MAEF108-crude agarases
| 與MA103-crude agarases 水解24 hr，經超過濾後總酚
| 含量、DPPH自由基之清除效應、及對亞鐵離子螯合效應
| 之變化    72
| 表6. 以 wound healing assay 定量12 與 24 hr細胞的遷移數量    73
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| 圖目錄
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| 圖1.    5% (w/v) 銀耳多醣熱萃液以MAEF108-crude agarases
| 與 MA103- crude agarases 水解24 hr，經超過濾後之
| 凝血酶原時間    74
| 圖2.    5% (w/v) 銀耳多醣熱萃液以MAEF108-crude agarases
| 與 MA103- crude agarases 水解24 hr，經超過濾後之
| 部分凝血活酶時間    75
| 圖3.  小鼠胚胎纖維母細胞之形態    76
| 圖4.  不同分子量區間與不同濃度之銀耳寡醣在10% FBS
| 下處理24 hr對3T3 細胞存活率的影響    77
| 圖5.  不同分子量區間與不同濃度之銀耳寡醣在10% FBS
| 下處理48 hr對3T3 細胞存活率的影響    78
| 圖6.  不同分子量區間與不同濃度之銀耳寡醣在10% FBS
| 下處理72 hr對3T3 細胞存活率的影響    79
| 圖7.  不同分子量區間與不同濃度之銀耳寡醣在1% FBS
| 下處理24 hr對3T3 細胞存活率的影響    80
| 圖8.  不同分子量區間與不同濃度之銀耳寡醣在1% FBS
| 下處理48 hr對3T3 細胞存活率的影響    81
| 圖9.  不同分子量區間與不同濃度之銀耳寡醣在1% FBS
| 下處理72 hr對3T3 細胞存活率的影響    82
| 圖10.    不同分子量區間之銀耳寡醣在含10% FBS之培養基中於
| 37oC 下培養48 hr後之膠原蛋白生成率    83
| 圖11.    不同分子量區間之銀耳寡醣在含1% FBS之培養基中於
| 37oC 下培養48 hr後之膠原蛋白生成率    84
| 圖12.    不同分子量區間之銀耳寡醣於1 mg/ mL濃度下處理48 hr
| 對於3T3細胞Collagen type I蛋白質之影響    85
| 圖13.    不同分子量區間之銀耳寡醣在含10% FBS之培養中於
| 37oC 下培養48 hr後之纖維結合蛋白含量    86
| 圖14.    三種分子量區間之銀耳寡醣之銀耳寡醣(1 mg/mL)
| 與2% TCA同時與3T3細胞反應4 hr後所呈現之保護作用    87
| 圖15.    三種分子量區間之銀耳寡醣之銀耳寡醣(1 mg/mL)
| 與2% TCA同時與3T3細胞反應4 hr後所呈現之修復作用    88
| 圖16.    倒立式顯微鏡放大100倍拍攝纖維母細胞於添加濃度
| 為1 mg/mL之銀耳寡醣(A)、(B)、(C)或(E)同時添加
| 銀耳寡醣與ERK抑制劑(E)或p38抑制劑(F)及(D)對照組
| 於37oC下培養48 hr，對增生之影響    89
| 圖17.    不同分子量區間之銀耳寡醣於1 mg/mL濃度下處理48 hr
| 對於3T3細胞 p-ERK 與 ERK 蛋白質之影響    90
| 圖18.    在倒立式顯微鏡40倍鏡頭下拍攝銀耳寡醣於1 mg/mL
| 濃度下，以wound-healing assay評估細胞於0、12、24
| 與48 hr後對小鼠纖維母細胞遷移行為之影響    91
| 圖19.        以wound healing assay評估添加 < 1 kDa之銀耳寡醣
| 於1 mg/ mL濃度下處理或同時添加ERK抑制劑或p38
| 抑制劑後12 hr對於3T3細胞collagen type I蛋白質之影響    92
| 圖20.    經創傷後以 < 1 kDa之銀耳寡醣或2 M PD98059與
| SB203580處理24 hr 後纖維結合蛋白含量    93
| 圖21.    經創傷後以 < 1 kDa之銀耳寡醣或2 M PD98059與
| SB203580處理24 hr 後TGF- 含量    94
| 圖22.    經創傷後以 < 1 kDa之銀耳寡醣或2 M PD98059與
| SB203580處理24 hr 後MMP-2 含量    95
| 圖23.    經創傷後以 < 1 kDa之銀耳寡醣或2 M PD98059與
| SB203580處理24 hr 後MMP-9 含量    96
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| 附錄
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| 附錄1.    銀耳子實體    97
| 附錄2.    皮膚的結構    98
| 附錄3.    傷口癒合的過程    99
| 附錄4.    膠原蛋白種類及分佈    100
| 附錄5.    第一型膠原蛋白的結構    101
| 附錄6.    經 1 mg/ mL，< 1 kDa 之銀耳寡醣或 ERK 抑制劑、p38
| 抑制劑處理 24 hr 後以倒立式顯微鏡放大 100 倍及 200
| 倍拍攝之纖維母細胞    102
| 附錄7.    比較含 10% FBS 培養 NIH-3T3 cell 並處理
| 不同濃度之銀耳寡醣於 48 hr 之細胞增生率    103
| 附錄8.    不同分子量區間之銀耳寡醣在含 1% 或 10% FBS 之
| 培養基中培養72 hr 之膠原蛋白生成率。    104
| 附錄9.    不同分子量區間之銀耳寡醣在含 10% FBS之
| 培養基，72 hr 纖維結合蛋白含量。    105
| 附錄10.    選擇 TCA 適合的濃度    106
| 附錄11.    三種分子量區間之銀耳寡醣對於 2% TCA 處理 4 hr 後
| 對3T3 細胞的保護作用    107
| 附錄12.    三種分子量區間之銀耳寡醣對於 2% TCA 處理 4 hr 後
| 對3T3 細胞的修復作用    108
| 附錄13.    以 wound healing assay 定量6 hr細胞的遷移數量    109
| 附錄14.    以 wound healing assay 定量48 hr細胞的遷移數量    110
| 附錄15.    不同分子量區間之銀耳寡醣在含10% FBS之培養基
| 處理48 hr後 TGF-之含量    111
| 附錄16.    經創傷後以 < 1 kDa 之銀耳寡醣或 2 M PD98059與
| SB203580 處理6、12、24與48 hr 後 TGF- 含量    112
| 附錄17.    經創傷後以 < 1 kDa 之銀耳寡醣或 2 M PD98059與
| SB203580處理6、12、24與48 hr 後Fibronectin含量    113
| 附錄18.    經創傷後以 < 1 kDa 之銀耳寡醣或 2 M PD98059與
| SB203580處理 48 hr 後 MMP-2 含量    114
| 附錄19.    經創傷後以 < 1 kDa 之銀耳寡醣或2 M PD98059與
| SB203580 處理 48 hr 後MMP-9 含量    115
| 附錄20.    凝固試驗流程圖    116
| 附錄21.    目前已知的 MMP 與其作用基質    117
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2010 年 3 月 8 日 22:49