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研究生:羅翠芬
研究生(外文):Tsue-Feng Lo
論文名稱:不同品系山藥貯存性蛋白質Dioscorin對人類呼吸道上皮細胞保護之研究
論文名稱(外文):An Evaluation of Protective Effect of Dioscorin from Various Chinese Yam Species on the Airway Epithelium Cells
指導教授:林國維林國維引用關係
指導教授(外文):Kuo-Wei Lin
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:80
中文關鍵詞:山藥儲存性蛋白質dioscorin肺癌細胞MTT流式細胞儀
外文關鍵詞:yamstuber storage proteinsdioscorinlung cancer cellsMTT assayflow cytometer
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山藥為中國傳統的養生藥膳。由山藥中純化出的儲存性蛋白質dioscorin,具有抗氧化及抗胰蛋白酶等活性。本文萃取與純化不同品種山藥(台農二號、基隆以及大汕)之dioscorin,鑑定抗氧化特性及熱穩定性。另外體外試驗再以呼吸道上皮細胞為材料測試dioscorin 在MTT試驗、細胞抗原反應及細胞週期的影響。結果顯示由三種山藥萃取出的dioscorin均有抗氧化活性,然三種彼此間均無顯著差別,並發現經過加熱 (100℃,1 min) 與未經過加熱的dioscorin組別,抗氧化性均沒有顯著的差異 (P>0.05),因此顯示dioscorin在此活性上有熱穩定性,其抗氧化性不因加熱而下降。
在MTT試驗中, A549細胞存活率隨著不同品種山藥dioscorin濃度增加有顯著下降的趨勢(80~60%),H460細胞存活率則隨著dioscorin濃度增加有些微下降的趨勢(95~85%),但針對正常白血球細胞則存活率介於 120~145%範圍,顯示並無毒殺作用。未加熱的dioscorin組別相較於加熱的組別其抑制細胞生長的活性較強,但隨加熱不同品種山藥之dioscorin濃度增加,A549及H460細胞存活率均分別有些微下降的趨勢 (95~80%,90~70%),推測 dioscorin對於A549與H460細胞有抑制生長的作用,且對於正常白血球細胞沒有毒殺的作用。經過加熱之dioscorin會降低其抑制A549與H460細胞生長的活性,但活性並未消失。經流式細胞儀觀察dioscorin對於A549細胞之細胞表面抗原的影響,發現dioscorin並不會引起發炎反應相關的細胞表面抗原增加。另外, dioscorin對於A549細胞並未發現明顯凋亡的情況。山藥之dioscorin濃度在1000 μg/ml以下會抑制A549與H460細胞生長,且不會毒殺正常白血球細胞,但是能否成為對抗肺癌的保健材料,還有待進一步的體內以及人體試驗來證實。
Chinese yam (Dioscorea spp). is one of Chinese traditional produces for consumption and medicinal purposes. Dioscorin, storage protein purified from yams showed multi-functions such as antioxidant and anti-trypsin activity. The objectives of this study were to investigate the antioxidant activity and heat stability of dioscorin from various yam species (Tainung No. 2, TN2; Keelung, KL; Tashan, TS). The effect of dioscorin on human airway cells in vitro was evaluated via MTT test, immunophenotyping and cell cycle. Similar antioxidant activity was found for unheated and heated (100 °C, 1 min) dioscorin from all species indicating thermostability of dioscorin.
The survival rate of A549 cells evaluated by MTT test showed a decreasing trend with ascending dioscorin levels of various yam cultivars, indicating toxicological effect of dioscorin on human pneumocyte A549 cells. The survival percentage of H460 cells decreased slightly as dioscorin concentrations increased. On the other hand, the survival percentage was relatively constant (120~145%) for normal white blood cells, suggesting no toxicological effect. Unheated dioscorin had higher inhibitory activity on cell growth than did heated dioscorin, but the cell survival percentage of A549 and H460 dropped slightly with increasing dioscorin levels. A possible inhibitory activity on the growth of A549 and H460 cells with no detrimental effect on normal white blood cells is proposed. Heating lowered dioscorin’s suppressive activity on cell growth, but this activity did not disappear regardless of heating. Results from the immunophenotyping of flow cytometer of A549 cells showed that addition of dioscorin didn’t induce inflamematory responses for the cell surface antigens. Additionally, cell cycle of A549 cells in response to dioscorin didn’t revral apparent apoptosis. In summary, dioscorin at present levels (100-1000 μg/ml) suppressed in vitro the growth of A549 and H460 cells without harming normal white blood cells. Potential utilization of dioscorin as a health agent is proposed; nonetheless, application of dioscorin into practical commercialized product as a protective media against lung cancer requires high scrutiny in vivo and human studies.
目錄...............................I
表目錄.............................Ⅲ
圖目錄.............................Ⅳ
中文摘要...........................Ⅵ
英文摘要...........................Ⅷ
第一章前言..........................1
第二章文獻回顧
第一節山藥..........................4
壹、品種及產地.......................4
貳、功效............................6
參、儲存性蛋白質.....................7
第二節天然中草藥抗癌.................10
第三節細胞週期......................12
第三章實驗目的......................15
第四章材料與方法
第一節實驗材料......................16
第二節實驗方法
壹、組成分分析......................18
貳、山藥儲存蛋白質純化...............22
參、抗氧化特性測定...................30
肆、細胞培養........................32
第五章結果
第一節山藥部分
壹、組成分分析......................37
貳、dioscorin之萃取與純化............37
參、抗氧化特性測定...................39
肆、相關性...........................45
第二節細胞培養
壹、MTT.............................45
貳、細胞型態.........................56
參、流式細胞儀.......................56
第六章討論...........................64
第七章結論...........................70
第八章參考文獻........................73
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