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研究生:林昱文
研究生(外文):Yu-Wen Lin
論文名稱:愛玉子種子水萃物癌症預防活性之體外試驗
論文名稱(外文):In vitro study on cancer-preventive activity of the water extract from jelly fig (Ficus awkeotsang Makino) seeds
指導教授:吳瑞碧
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2008
畢業學年度:97
語文別:中文
論文頁數:166
中文關鍵詞:愛玉子種子DNA保護氧化應力細胞凋亡抗癌癌細胞
外文關鍵詞:jelly fig seedsDNA protectionoxidative stressapoptosisantitumorcancer cell
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愛玉子為台灣地區特有的富含果膠之木質藤本植物種子,分布在海拔800至1800公尺之間的山區。本研究將愛玉子種子,以果汁機粉碎,加水萃取的方式,得到含有可溶性固形物的水萃液,再經由冷凍乾燥,獲得愛玉子種子水萃物 (water extract from jelly fig seeds, WEJFS),為試驗原料。
利用體外 (in vitro) 試驗探討愛玉子種子水萃物的細胞毒性、保護牛胸腺細胞DNA、大鼠肝臟clone 9細胞的DNA和Clone 9細胞的作用,以及對小鼠大腸癌細胞CT-26、人類肝癌細胞HepG2和人類雌激素依賴型乳癌細胞MCF-7生長之影響,並且分析硫酸銨和膜區分 (fraction) 成分與癌細胞生長抑制的關係。研究結果顯示每毫升培養液含有0至200 μg的愛玉子種子水萃物無細胞毒性。愛玉子種子水萃物對DNA和Clone 9細胞具有保護作用,包括降低氫氧自由基誘導的DNA損傷,與抑制過氧化氫造成Clone 9細胞的死亡。在水萃物保護Clone 9細胞之測定結果顯示提高細胞的存活率,降低Clone 9細胞的caspase-3、亞雙倍體比率,並阻止DNA產生片段化。愛玉子種子水萃物對於小鼠大腸癌細胞CT-26、人類肝癌細胞HepG2和人類雌激素依賴型乳癌細胞MCF-7皆有生長抑制的效果,且以抑制CT-26和HepG2細胞的生長能力較佳。使用截留分子量 (molecular weight cut off, MWCO) 為0.5、3.0、10.0及70.0 kDa的過濾膜過濾所獲得的膜區分水萃物,以3.0至10.0 kDa膜區分物對HepG2細胞具有較高的生長抑制能力。此3.0至10.0 kDa膜區分水萃物在三甲基甘胺酸胜肽電泳表現7 kDa的主要色帶,並無洋刀豆血球凝集素A親和式膠體鍵結的能力。利用不同比例的飽和硫酸銨分劃與HepG2細胞培養,結果發現40至80%的分劃對細胞具有生長抑制能力,在此比例的硫酸銨分劃中,是以蛋白質為主要的化學成分。以上各試驗所呈現的結果,顯示愛玉子種子水萃物潛在具有癌症之化學預防活性。
Jelly fig (Ficus awkeotsang Makino) achenes are the seeds of a native woody vine that grows on 800-1800 m high hillsides in Taiwan. In the present study, a water extract from jelly fig seeds (WEJFS) was prepared to investigate the cytotoxicity on rat hepatocyte clone 9 cells, and the protective effect on clone 9 cells and their DNA against oxidative stress damage. The growth inhibition on three cancer cell lines, namely mouse colon cancer CT-26, human hepatoma HepG2 and breast cancer MCF-7 were also studied. Components from ultrafiltration and ammonium sulfate fractionations were analyzed for their activities on the growth inhibition of HepG2 cancer cells.
Results showed that WEJFS at 0-200 μg total solids/mL concentration had no cytotoxicity on Clone 9 cells, and that WEJFS inhibited the hydrogen peroxide induced damage in Clone 9 cells as shown by the caspase-3 activity, SubG1 (%), and DNA fragmentation. The anticancer activities on the above-mentioned cancer cell lines were also shown. Among them, CT-26 and HepG2 cells were inhibited more effectively. Filtrates of ultrafiltration within different membrane cut-off ranges were used to test the activity of growth inhibition from WEJFS. The 3.0-10.0 kDa fraction exhibited the highest inhibition ability on HepG2 cells. SDS-PAGE of the fraction found a major band at 7 kDa. The 3.0-10.0 kDa fraction was found without concanavalin A sepharose affinity column binding ability. Among the saturated ammonium sulfate precipitation fractions, the 40-80% fraction showed the highest growth inhibition ability on HepG2 cells. Protein was found to be the major component in the fraction. In conclusion, these results suggested that WEJFS can be a decent potential agent for chemoprevention of cancers.
摘 要 ..………………….....……..……..…………………….…...Ⅰ
ABSTRACT .…………………………………………..…….….…...Ⅲ
目 錄 ……………………………………..……………….….…....Ⅴ
圖 次 ...………………………………………………………..…..ⅩⅡ
表 次 ...………………………………………………………….…ⅩⅤ
前 言 ………………………………………..………………......…..1
第一章 文獻整理 ……………..……………………………..….….3
第一節 氧化應力與防禦 ………...…………………………….……….4
1.1.1反應性氧和氮分子的形成 …………………...……………..…….4
1.1.2氧化應力和對成分的傷害 …...……………………………..……5
1.1.3氧化應力防禦 ……...……………………………………..………6
第二節 癌症的發生與預防 ...……………………………….…..…….12
1.1.1誘發癌症之危險因子 .…………….…………………....…..……12
1.1.2腫瘤形成的步驟 .......................................................................12
1.1.2.1起始期 ………………………………………………….……..12
1.1.2.2促進期 ………………………………………………….……..13
1.1.2.3發展期 ………………………………………………….……..13
1.1.3癌症預防與癌症化學預防 ………………...………………….……13
1.1.3.1癌症的預防 ……………………………………………….….13
1.1.3.2癌症的化學預防劑 ……………………………….………….14
(1) 致癌物質抑制劑 .....................................................................14
(2) 癌症阻斷劑 ………………………………………….………….14
(3) 癌症抑制劑 …………………………………………….……….15
1.1.3.3癌症化學預防的方法 ………………………………………..15
1.1.4天然物中的化學預防劑與作用機制 …………………………..16
1.1.4.1天然物中的化學預防劑 ……………………………………..16
1.1.4.2天然物中化學預防劑的作用機制 ………………………….16
(1) 解毒酵素的調節作用 ………………………………………….16
(2) 細胞週期停滯 ………………………………………….………18
(3) 細胞凋亡 ………………………………………………….…….19
第三節 愛玉子特性和功能 ………………………………………….33
1.3.1愛玉子由來 ………………………………………………………33
1.3.2愛玉子的成分和構造 …………………………………………...33
1.3.3愛玉子的功能研究 ……………………………………………...34
第二章 實驗設計與架構 …………………………………………37
第一節 實驗架構 …………………………………………….……….38
第二節 實驗設計 …………………………………………….……….38
2.2.1實驗Ⅰ (ExpⅠ) ………………………………………………….38
2.2.2實驗Ⅱ (ExpⅡ) ………………………………………………….38
2.2.3實驗Ⅲ (ExpⅢ) ………………………………………………….39
第三章 愛玉子種子水萃物的成分和細胞毒性探討 …………...41
第一節 摘 要 ………………………………………………………...42
第二節 實驗材料 …………………………………………………….43
3.2.1實驗原料 ………………………………………………………...43
3.2.2實驗細胞 ………………………………………………………...43
3.2.3實驗藥品 ………………………………………………………...43
3.2.4實驗藥品配製 …………………………………………………...45
3.2.5實驗材料與儀器 ...............................................................................45
第三節 實驗方法 ............................................................................48
3.3.1愛玉子種子水萃物製備 ...........................................................48
3.3.2愛玉子種子水萃物的成分分析 ................................................48
3.3.2.1碳水化合物 ..........................................................................48
3.3.2.2多酚類化合物 ......................................................................48
3.3.2.3蛋白質 .................................................................................49
3.3.3細胞株解凍、繼代培養和冷凍 ………………………………..49
3.3.3.1細胞株解凍 …..………………………………………………49
3.3.3.2細胞株的繼代培養 …..………………………………………49
3.3.3.3細胞株冷凍 …………..………………………………………50
3.3.4細胞株計數法 …………………………………………………...50
3.3.5試驗樣品處理和細胞存活率測定 ...........................................51
3.3.5.1測試樣品處理 ………………………………………………..51
3.3.5.2細胞株存活率 ………………………………………………..51
3.3.6統計分析 ………………………………………………………...52
第四節 結果與討論 ........................................................................57
3.4.1愛玉子種子水萃物的成分 .......................................................57
3.4.2愛玉子種子水萃物的細胞毒性 ...............................................58
第五節 結 論 ................................................................................66
第四章 愛玉子種子水萃物預防氧化應力誘導損傷之預防 ………67
第一節 摘 要 ................................................................................68
第二節 實驗材料 …………………………………………………….69
4.2.1實驗原料 …………………………………………………………69
4.2.2實驗細胞 …………………………………………………………69
4.2.3實驗藥品 …………………………………………………………69
4.2.4實驗藥品配製 …………………………………………………...71
4.2.5實驗材料與儀器 ...............................................................................72
第三節 實驗方法 ............................................................................75
4.3.1愛玉子種子水萃物製備 ...........................................................75
4.3.2細胞株冷凍、解凍和繼代培養 ………………………………..75
4.3.3細胞株計數法 …………………………………………………...75
4.3.4試驗樣品處理和細胞存活率測定 ...........................................75
4.3.4.1測試樣品處理 ………………………………………………..75
4.3.4.2細胞存活率 …………………………………………………..75
4.3.5氫氧自由基誘導的DNA損傷 …………………………………76
4.3.5.1萃取細胞的DNA ……………………………………………..76
4.3.5.2評估氫氧自由誘導的DNA損傷 ……………………………76
4.3.6細胞DNA的片段化 …………………………………………….77
4.3.7細胞的週期分布 ………………………………………………...77
4.3.8細胞凋亡蛋白caspase-3的活性分析 ………………………….78
4.3.9統計分析 ………………………………………………………...79
第四節 結果與討論 .........................................................................80
4.4.1愛玉子種子水萃物降低氫氧自由基誘導的DNA損傷 ..........80
4.4.2愛玉子種子水萃物對抗過氧化氫誘導的細胞毒性 ................80
4.4.3愛玉子種子水萃物抑制過氧化氫誘導的caspase-3活性 .......82
4.4.4愛玉子種子水萃物影響過氧化氫產生的細胞週期分布 .........83
4.4.5愛玉子種子水萃物阻止過氧化氫誘導的DNA片段化 ..........85
第五節 結 論 ……………………………………………………….99
第五章 愛玉子種子水萃物的抑癌作用和不同區分的成分探討
…………...………………………………………………100
第一節 摘 要 ………………………………………….…………..101
第二節 實驗材料 …………………………………………………...103
5.2.1實驗原料 ……………………………………………………….103
5.2.2實驗細胞 ………………………………………………………….103
5.2.2.1人類肝癌細胞株 ……………………………………………103
5.2.2.2小鼠大腸癌細胞株 ………………………………….….......103
5.2.2.3人類雌激素依賴型乳癌細胞株 …………………………...103
5.2.3實驗藥品 ……………………………………………………….103
5.2.3.1實驗藥品試劑 ……………………………………………....103
5.2.4實驗藥品配製 ………………………………………………….106
5.2.5實驗材料與儀器 …………………………………….……….…...107
第三節 實驗方法 …………………………………………………..….111
5.3.1愛玉子種子水萃物製備 ……………………………...………..111
5.3.2膜區分水萃物之製備 ………………………………………….111
5.3.3硫酸銨分劃溶液之製備 ……………………………………….111
5.3.4成分分析 ………………………………………………….….…112
5.3.4.1碳水化合物 ........................................................................112
5.3.4.2多酚類化合物 ....................................................................112
5.3.4.3蛋白質 ...............................................................................112
5.3.4.4胜肽濃度測定 ……………………………………………....112
5.3.5癌細胞株之繼代培養、冷凍與解凍 …………………………….113
5.3.6 細胞計數法 ……………………………………………………....113
5.3.7試驗樣品處理和癌細胞生長抑制率 ……………….…………113
5.3.7.1測試樣品處理 …………………………………….…………113
5.3.7.2癌細胞生長抑制率 ……………………………………..…..113
5.3.8 高效率液相層析 ………………………………………………113
5.3.9 三甲基甘胺酸-SDS膠體電泳分析 …………………………..114
5.3.9.1鑄膠 ………………………………………………………….114
3.5.9.2樣品製備 ……………………………………………….……115
5.3.9.3電泳操作 ……………………………………………….……115
5.3.9.4膠片染色 ……………………………………………….……115
5.3.9.5膠片掃描 ………………………………………………….…115
5.3.10洋刀豆血球凝集素A膠體層析 ……………………….….…115
5.3.11統計分析 ……………………………………………….….…..116
第四節 結果與討論 …………………………………………….……..120
5.4.1 愛玉子種子水萃物對不同癌細胞的生長抑制 …………………120
5.4.2 膜區分水萃物對HepG2的生長抑制 …………………………..122
5.4.3 膜區分水萃物的化學成分和特性 ……………………….…..122
5.4.3.1愛玉子膜區分物的化學成分 ………………………………122
5.4.3.2膜區分的成分特性 …………………………………………124
5.4.4 硫酸銨分劃對HepG2的生長抑制和成分特性 …………….125
5.4.4.1硫酸銨分劃對HepG2的生長抑制 …………………………125
5.4.4.2硫酸銨分劃的成分特性 ……………………………….…….126
第五節 結 論 ………………………………………………………...145
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