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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張鴻民 | |
dc.contributor.author | Da-Wei Huang | en |
dc.contributor.author | 黃大維 | zh_TW |
dc.date.accessioned | 2021-06-13T07:51:35Z | - |
dc.date.available | 2008-07-28 | |
dc.date.copyright | 2005-07-28 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-25 | |
dc.identifier.citation | 參 考 文 獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36110 | - |
dc.description.abstract | 摘要
雜環胺 ( Heterocyclic amines;HCAs ) 已被證實為高度致突變及致癌物質,而Trp-P-1為HCAs中毒性較強的種類之一。HCAs之毒性是經細胞色素 cytochrome P-450之活化後產生。本實驗是以蛋白質體學來研究以Trp-P-1處理人類肝癌細胞株 HepG2對其蛋白質表現之影響。細胞實驗結果顯示,Trp-P-1處理濃度大於2 µg/mL 時生長抑制率會隨天數增加而有明顯上升之趨勢,且以3、4、5 µg/mL Trp-P-1處理至第三天後,其生長抑制率已達90 % 以上。蛋白質體學分析顯示,HepG2以2 µg/mL Trp-P-1處理1、2、3天,蛋白質表現量增加且有50 % 以上差異者於第1天有6 個點、第2天有9個點、第3天有11個點;而表現量減少且有50 % 以上差異者於第2天有3個點、第3天有4個點。將這些表現量差異性大於50 % 之蛋白質點以胰蛋白酶 ( trypsin ) 進行膠內水解後以基質輔助雷射吸附游離離子化飛行時間 ( MALDI-Q-TOF ) 質譜儀進行分析,並經資料庫的比對以鑑定出蛋白質的種類及功能。結果在第1天處理組中有4個點被鑑定出;第2天處理組中有10個點被鑑定出;第3天處理組中有13個點被鑑定出,這些蛋白質功能分析顯示,Trp-P-1處理所造成的細胞生理變化主要以能量代謝循環及維持蛋白質結構為主。 | zh_TW |
dc.description.abstract | Abstract
Heterocyclic amines have been proved to be highly mutagenic and carcinogenic due to the activation of cytochrome P-450 in vivo. Among them, Trp-P-1 is one of the potent toxic heterocyclic amines. In the present study, proteomics on the protein expression of HepG2 cells following treatment with Trp-P-1 were conducted. In vitro, the growth inhibition of HepG2 cells increased in a time-dependent manner after treatment with Trp-P-1 at a level higher than 2 µg/mL. More than 90 % cells were inhibited when treated with 3, 4 or 5 µg/mL Trp-P-1 for 3 days. In proteomic analysis, 6, 9, and 11 protein spots were observed to be increased in quantity by more than 50 % if HepG2 cells were treated with 2 µg/mL Trp-P-1 for 1, 2, and 3 days, respectively. However, protein expression decreased in quantity by more than 50 % were determined to be 3 and 4 protein spots treated with the same amount of Trp-P-1 for 2 and 3 days, respectively. These protein spots were in gel digested by trypsin and analyzed by matrix assisted laser desorption ionization – time of flight ( MALDI-TOF ) mass spectrometry and 4, 10, and 13 proteins were successfully identified in day-1, -2, and -3 sample, respectively. Internet database ( Swiss-Prot ) search indicated that the changes in cell physiology induced by Trp-P-1 were mainly due to the influence of protein functions on energy metabolism cycle and protein structure maintain. | en |
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dc.description.tableofcontents | 目 錄
前言…………………………………………………………………….-1- 第一章 文 獻 回 顧…………………………………………….......-2- 一、雜環胺化合物 (一) 雜環胺化合物簡介………………………………………...-2- ( 1 ) 由前驅物合成的 HCAs…………………………….....-2- ( 2 ) 直接由胺基酸或蛋白質經熱裂解生成的 HCAs…….-3- (二) HCAs 之分類…………………………………………........-3- ( 1 ) aminoimidazo-quinoxaline HCAs 衍生物…………......-3- ( 2 ) aminoimidazo-quinoline HCAs 衍生物…………….....-4- ( 3 ) pyrido HCAs 衍生物…………………………………..-4- ( 4 ) pyrido-indole HCAs 衍生物……………………….......-4- (三) HCAs的生物毒性…………………………………………-4- (四) Trp-P-1之介紹…………………………………………....-10- (五) 影響食品中HCAs 生成之因素………………………....-10- ( 1 ) 加熱溫度對HCAs 生成之影響……………………..-10- ( 2 ) 加熱時間對 HCAs 生成之影響………………….....-11- ( 3 ) 加熱方式對 HCAs 生成之影響………………….....-11- ( 4 ) 食品中水含量對 HCAs 生成的影響…………….....-12- ( 5 ) 食品中脂肪含量對 HCAs 生成的影響………….....-12- ( 6 ) 食品中抗氧化劑對 HCAs 生成的影響………….....-13- 二、蛋白質體之研究…………………………………………….-14- (一) 蛋白質體 ( proteome )……………………………….......-14- (二) 蛋白質體學 ( proteomics )………………………………-15- (三) 蛋白質體學的種類………………………………………-16- ( 1 ) 表現蛋白質體學 ( expression proteomics )………...-16- ( 2 ) 功能蛋白質體學 ( functional proteomics )…………-16- ( 3 ) 結構蛋白質體學 ( structural proteomics )………….-16- (四) 蛋白質體學技術…………………………………………-17- ( 1 ) 樣品之前處理………………………………………..-17- ( 2 ) 二維電泳 ( two-dimensional gel electrophoresis ; 2D-GE )……………………………………………...-18- ( 3 ) 蛋白質顯色…………………………………………..-19- ( 4 ) 質譜 ( mass spectrometry,MS ) 分析……………..-21- ( 5 ) 資料庫比對…………………………………………..-22- 第二章 材 料 與 方 法…………………………………………..-24- 第一部份:Trp-P-1對人類肝癌細胞株 HepG2 之影響………...-24- 一、實驗樣品……………………………………………………-24- 二、實驗細胞……………………………………………………-24- 三、實驗藥品與試劑……………………………………………-24- 四、實驗器材……………………………………………………-25- 五、儀器設備……………………………………………………-25- 六、溶液配置……………………………………………………-26- 七、樣品測試之制備……………………………………………-27- 八、HepG2細胞冷凍、解凍及繼代培養……………………....-27- 九、細胞計數……………………………………………………-28- 十、細胞存活率測定- MTT assay………………………………-29- 十一、細胞生長抑制率之計算…………………………………-30- 十二、統計分析…………………………………………………-30- 第二部份:以蛋白質體學分析Trp-P-1處理人類肝癌細胞株 HepG2………………………………………………...-32- 一、實驗樣品……………………………………………………-32- 二、實驗細胞……………………………………………………-32- 三、實驗藥品與試劑……………………………………………-32- 四、實驗器材……………………………………………………-33- 五、儀器設備……………………………………………………-33- 六、溶液配置……………………………………………………-34- 七、蛋白質體分析HepG2之蛋白質製備……………………...-38- 八、HepG2蛋白質定量………………………………………....-39- 九、迷你電泳分析……………………………………………….-41- (一) 鑄膠………………………………………………………-41- (二) 樣品製備…………………………………………………-42- (三) 電泳操作…………………………………………………-42- (五) 膠片乾燥…………………………………………………-42- 十、二維式電泳………………………………………………….-43- (一) 第一維電泳……………………………………………...-43- (二) 第二維電泳……………………………………………...-43- (三) 膠片染色………………………………………………...-44- (四) 膠片掃描………………………………………………...-44- (五) 影像分析比對…………………………………………...-44- 十一、蛋白質鑑定……………………………………………….-45- (一) 蛋白質膠內水解 ( in-gel digestion )……………………-45- (二) 質譜分析…………………………………………………-46- (三) 資料庫檢索………………………………………………-47- 第三章 結 果 討 論……………………………………………….-48- 一、Trp-P-1對人類肝癌細胞株 HepG2 之影響……………....-48- (一) 生長抑制率之比較………………………………….....-48- (二) 細胞型態觀察……………………………………….....-51- 二、以蛋白質體學分析Trp-P-1處理人類肝癌細胞株 HepG2…………………………………………………........-55- (一) 電泳檢定法………………………………………….....-55- (二) 二維式電泳分析……………………………………… -58- (三) 電泳膠片影像比對分析……………………………….-61- (四) 蛋白質質譜分析鑑定………………………………….-64- (五) 酵素性蛋白質之探討………………………………….-81- 第四章 總結………………………………………………………….-89- 第五章 參 考 文 獻………………………………………………...-90- 圖 次 圖2-1、以MTT法所得之HepG2細胞標準曲線…………………..-31- 圖2-2、牛血清蛋白以Bio-Rad蛋白質定量法所得的標準曲線…..-40- 圖3-1、不同濃度Trp-P-1處理HepG2細胞生長抑制率之比較…..-50- 圖3-2、0.5 µg/mL Trp-P-1於不同天數對HepG2細胞形態之觀察……………………………………………………………-52- 圖3-3、1 µg/mL Trp-P-1於不同天數對HepG2細胞形態之觀察…-52- 圖3-4、2 µg/mL Trp-P-1於不同天數對HepG2細胞形態之觀察....-53- 圖3-5、3 µg/mL Trp-P-1於不同天數對HepG2細胞形態之觀察....-53- 圖3-6、4 µg/mL Trp-P-1於不同天數對HepG2細胞形態之觀察....-54- 圖3-7、5 µg/mL Trp-P-1於不同天數對HepG2細胞形態之觀察....-54- 圖3-8、以2 µg/mL Trp-P-1處理HepG2細胞經CBR染色之SDS- PAGE膠片圖……………………………………………….-57- 圖3-9、不同等電點焦集條件下得到之結果 ( A ) 未焦集 ; ( B ) 焦集 ; ( C ) 過焦集………………………………………….-60- 圖3-10、以2 µg/mL Trp-P-1處理HepG2細胞之細胞蛋白質二維電泳圖 ( A ) 控制組 ( 0.5 % DMSO ) ; ( B ) 1天 ; ( C ) 2天 ; ( D ) 3天……………………………………………………-63- 圖3-11、HepG2以2 μg/mL Trp-P-1處理1天之二維電泳圖……..-66- 圖3-12、HepG2以2 μg/mL Trp-P-1處理1天之蛋白質表現 ( upregulated proteins )……………………………………-67- 圖3-13、HepG2以2 μg/mL Trp-P-1處理2天之二維電泳圖……..-69 - 圖3-14、HepG2以2 μg/mL Trp-P-1處理2天之蛋白質表現 ( downregulated proteins )………………………………....-70- 圖3-15、HepG2以2 μg/mL Trp-P-1處理2天之蛋白質表現 ( upregulated proteins )…………………………………….-71- 圖3-16、HepG2以2 μg/mL Trp-P-1處理3天之二維電泳圖……..-74- 圖3-17、HepG2以2 μg/mL Trp-P-1處理3天之蛋白質表現 ( downregulated proteins )………………………………....-75- 圖3-18、HepG2以2 μg/mL Trp-P-1處理3天之蛋白質表現 ( upregulated proteins )…………………………………….-76- 圖3-19、醣解及糖質新生作用………………………………………-82- 圖3-20、果醣及木糖之代謝途徑…………………………………....-83- 圖3-21、脂肪酸之代謝途徑…………………………………………-84- 圖3-22、丙酮酸鹽之代謝途徑……………………………………....-85- 圖3-23、肌醇之代謝途徑……………………………………………-86- 圖3-24、ATP合成作用………………………………………………-87- 表 次 表1-1、雜環胺之致突變分析………………………………………...-6- 表1-2、老鼠試驗中 HCAs 會影響之器官………………………….-8- 表1-3、HCAs 對老鼠所測得的 TD50 值……………………………-9- 表3-1、不同濃度Trp-P-1處理HepG2細胞生長抑制之比較……..-49- 表3-2、蛋白質特性及功能 ( Trp-P-1 處理1天)…………………..-68- 表3-3、蛋白質特性及功能 ( Trp-P-1 處理2天)…………………..-72- 表3-4、蛋白質特性及功能 ( Trp-P-1 處理3天)…………………..-77- 表3-5、經Trp-P-1處理後蛋白質表現之鑑定……………………...-79- | |
dc.language.iso | zh-TW | |
dc.title | 以蛋白質體學探討雜環胺Trp-P-1處理人類肝癌細胞株HepG2 | zh_TW |
dc.title | Proteomic Analysis of Human Hepatoma G2 Cells Following Treatment with Heterocyclic Amines Trp-P-1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳水田,陳炳輝,張基郁,張雲祥 | |
dc.subject.keyword | 雜環胺, 人類肝癌細胞株, 蛋白質體學, 基質輔助雷射吸附游離離子化飛行時間質譜儀, | zh_TW |
dc.subject.keyword | Heterocyclic amines, HepG2, Proteomics, MALDI-Q-TOF, | en |
dc.relation.page | 101 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-25 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 食品科技研究所 | zh_TW |
顯示於系所單位: | 食品科技研究所 |
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