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研究生:柯婉茹
研究生(外文):Wan-JuKo
論文名稱:雌激素探針之製備及於化學蛋白質體學之應用
論文名稱(外文):Fabrication of estrogen probes for chemical proteomics
指導教授:陳淑慧陳淑慧引用關係
指導教授(外文):Shu-Hui Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:74
中文關鍵詞:雌激素金奈米粒子生物素
外文關鍵詞:estrogengold nanoparticlesbiotin
相關次數:
  • 被引用被引用:1
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  • 下載下載:4
  • 收藏至我的研究室書目清單書目收藏:0
乳癌的發生率在全世界都非常高,尤其是女性獲得乳癌的機率也相對較男性來的高,衛生署去年度的統計資料,女性乳癌的排名位居第四,而雌激素不只是對乳房細胞有影響,對骨頭及泌尿系統也會有作用,當然最明顯的影響就是對上乳房細胞會有促進分化生長的癌化現象,所以如何利用金奈米粒子載體,針對乳癌細胞中的雌激素受體進行純化濃縮,將在後續的實驗對於雌激素探針和受體進行探討。
利用金奈米粒子作為固態載體,並配合類固醇的雌激素小分子 (E2-biotin),利用 biotin-avidin 系統,對乳癌細胞 (MCF-7) 細胞萃取液中的雌激素受體蛋白的其中一個構型 (ERα) 進行親和性作用而被金奈米粒子純化。在實驗當中以含有 E2-biotin 作為實驗組,控制組則是不含雌激素衍生物,利用非直接修飾在奈米金粒子上的雌激素衍生物,降低系統中的非專一性吸附蛋白,並將得到的目標蛋白經過酵素消化後,利用高壓液相層析質譜儀,辨認與雌激素受體相關的蛋白質,再經由蛋白質資料庫比對胜肽序列,提供在化學蛋白質體學上的一個新視野。

The most common cancer affecting women is breast cancer. Estrogens regulate multiple cellular function via estrogen receptor alpha (ERα). In order to have more information about various biological events by the estrogen binding position and surface character with ERα, this study performed an strategy to fabricate 17α-estradiol (E2) probe using gold nanoparticles (AuNP) in breast cancer cells. The positive AuNP probe contain E2-derivated incubate whole cell lysate, and negative AuNP probes were functionalized with polyethylene glycol (PEG) without E2-derivated. Applied this approach reducing the nonspecific binding of E2-derivated related to ERα, at the same time this approach following liquid chromatography tandem mass spectrometry to identify ERα related proteins. The indirect E2-derivated approach pulldown might give a new view on chemical proteomics approach in ERα related protein by LC-MS/MS and present an easy and repeatable experiment.
目錄
中文摘要 i
英文摘要 ii
圖目錄 vi
表目錄 ix
第一章 緒論 1
1.1 蛋白質體學 1
1.1.1質譜學 4
1.1.2 蛋白質體與質譜 13
1.2 雌激素與雌激素受體 15
1.3 奈米科技在生物醫學及鑑測的應用 19
1.3.1 金奈米粒子 19
第二章 實驗 22
2.1 藥品 22
2.2 儀器 22
2.3 奈米金探針 23
2.3.1 製備球狀金奈米粒子 23
2.3.2製備雌激素結合生物素小分子探針 (E2-biotin probe) 23
2.3.3 卵蛋白修飾金奈米探針 (Avidin-gold nanoparticle) 23
2.4 乳癌細胞培養 24
2.5 細胞萃取 24
2.5.1 雌激素修飾探針親和性純化全細胞萃取液 25
2.6 蛋白質染色法 25
2.6.1 西方點墨法 (Western bloting) 25
英文摘要 i
中文摘要 ii
致謝 iii
圖目錄 vi
表目錄 ix
第一章 緒論 1
1.1 蛋白質體學 1
1.1.1質譜學 4
1.1.2 蛋白質體與質譜 13
1.2 雌激素與雌激素受體 15
1.3 奈米科技在生物醫學及鑑測的應用 19
1.3.1 金奈米粒子 19
第二章 實驗 22
2.1 藥品 22
2.2 儀器 22
2.3 奈米金探針 23
2.3.1 製備球狀金奈米粒子 23
2.3.2製備雌激素結合生物素小分子探針 (E2-biotin probe) 23
2.3.3 卵蛋白修飾金奈米探針 (Avidin-gold nanoparticle) 23
2.4 乳癌細胞培養 24
2.5 細胞萃取 24
2.5.1 雌激素修飾探針親和性純化全細胞萃取液 25
2.6 蛋白質染色法 25
2.6.1 西方點墨法 (Western bloting) 25
2.6.2 銀染 (Silver stain) 26
2.6.3 膠體螢光染色 (SYPRO®Ruby protein gel stain) 26
2.7 三氯乙酸蛋白質沉澱法 (Trichloroacetic acid protein precipitation, TCA) 27
2.8 胜肽質譜分析數據處理 28
第三章 實驗結果與討論 29
3.1卵蛋白修飾金奈米粒子UV-VIS 吸收光譜 29
3.2 雌激素探針 31
3.3 E2-biotin 的分離純化對於雌激素受體的影響 33
3.4 雌激素受體重組蛋白 (rERα) 在基質內的表現 42
3.5 最佳化洗滌卵蛋白金奈米粒子溶液 46
圖27:不同洗滌溶液在不同體積下,對卵蛋白修飾金奈米粒子的非專一性吸附圖。 47
3.6 卵蛋白修飾金奈米粒子與市售膠體 48
3.7 質譜分析雌激素探針純化後的雌激素受體 50
第四章 結論 51
參考文獻 52
附錄 59


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