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研究生:潘俊佑
研究生(外文):Jun-yu Pan
論文名稱:合成雌激素金奈米探針應用於配位受體之親合力分析
論文名稱(外文):Estrogen Chemical Probe Fabricated on Au Nanoparticles for Ligand-Receptor Interaction
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:奈米科技暨微系統工程研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:108
中文關鍵詞:金奈米粒子雌激素雌激素受體
外文關鍵詞:avidinestrogen receptorestrogenbiotingold nanoparticles
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雌激素在人體的生長發育以及乳腺的變異扮演著重要的角色,研究也指出了內生的雌激素和乳癌及婦科的癌症息息相關。其中這些癌細胞的增生和擴散是藉著雌激素受體進行訊號的傳導且當作轉錄因子來對目標基因進行調控。而雌激素受體分為兩種型態,都是屬於核受體的家族,分別是alpha, beta,其中雌激素受體alpha更確切的被認為和腫瘤癌細胞的變異有關。活性化的雌激素受體alpha在乳癌中更是扮演著非常重要的角色,也是藥物治療的主要目標,因此本論文建立了一套平台能夠有效率地捕捉乳癌細胞中的活性化雌激素受體alpha,並應用於雌激素配位受體的親和力分析。
首先在本文的研究中利用了雌激素衍生物標定上biotin來對雌激素受體進行活化及結合,接著利用金奈米粒子其本身具有高的比表面積以及良好的生物相容性,經由表面化學修飾後接上avidin來當作探針,最後加上biotin-avidin系統之間非常強的親和力,降低了非特異性吸附及排除立體障礙,我們成功的達成目標,有效率地捕捉到活性化雌激素受體alpha,接著便應用於配位受體(雌激素,雌激素受體衍生物,及類固醇藥物)的親和力分析。
最後從結果顯示,我們建立了一個快速且能被廣泛應用的生物分析平台。
Estrogen plays essential roles in the development, growth control and differentiation of the normal mammary gland. However, it is well documented that endogenous estrogens are powerful mitogens critical for the initiation and progression of human breast and gynecological cancers. This cell proliferation signal is mediated by the estrogen receptors (ER), members of the nuclear receptor family that function both as signal transducers and transcription factors to modulate expression of target genes. There are two main subtypes of estrogen receptors: ERα and ERβ. ERα is certainly associated with more differentiated tumors.
Activated estrogen receptor alpha (ERα) plays a critical role in breast cancer development and is a major target for drug treatment, so our aim is to build up a high efficient platform which is able to pull down activated estrogen receptor (ERα) in MCF7 breast cancer cell and apply it to Ligand-Receptor interaction analysis.
In the article, we tag estrogen derivatives with biotin and use it to activate and pull down estrogen receptor. After that Au-NP is taken as a probe because of its large surface to volume ratio and low level of toxicity when introduced into biological systems. By surface chemistry, we conjugate Au-NP with avidin. Finally we successfully reduce non-specific binding and steric hindrance by using biotin-avidin system because of their highly strong affinity.
Upon this powerful pull down method established, we use it to analyze Ligand-Receptor interaction including estrogen, estrogen derivatives, and estrogenic drugs. Results indicate that we have developed a sufficiently convenient and high-throughput analysis probe for estrogen receptor binding of ligands and interaction determination.
第一章 序論
1-1 生醫奈米---------------------------------------------14
1-2 蛋白質體學-------------------------------------------16
1-3 雌激素作用模式---------------------------------------17
第二章 免疫螢光法分析雌激素及其受體於MCF-7細胞之分佈
2-1螢光抗體的原理---------------------------------------18
2-2螢光抗體法的特性-------------------------------------19
2-3實驗部份---------------------------------------------20
2-3-1 藥品與儀器------------------------------------21
2-3-2 免疫螢光法------------------------------------21
2-4結果與討論-------------------------------------------23
2-5結論-------------------------------------------------25
第三章 雌激素金奈米探針之合成
3-1 金奈米粒子------------------------------------------42
3-2 金奈米粒子與蛋白質的結合方法------------------------43
3-3 biotin-avidin 系統----------------------------------45
3-4雌激素衍生物之合成----------------------------------46
3-5 實驗部份--------------------------------------------47
3-5-1 藥品與儀器------------------------------------47
3-5-2 金奈米粒子的製備------------------------------48
3-5-3 biotinylate 雌激素衍生物----------------------48
3-5-4 金奈米粒子的修飾------------------------------49
3-6 結果與討論 -----------------------------------------50
3-6-1 UV及SEM鑑定金奈米粒子------------------------50
3-6-2 純化方法比較----------------------------------51
第四章 雌激素金奈米探針於親和力pull down之應用
4-1 親和性作用力----------------------------------------66
4-2 實驗部份--------------------------------------------67
4-2-1 藥品與儀器------------------------------------67
4-2-2 膠體的製備------------------------------------69
4-2-3 分離樣品--------------------------------------69
4-2-4 膠體染色--------------------------------------70
4-2-5 膠體內的消化----------------------------------70
4-2-6 質譜分析--------------------------------------71
4-2-7 免疫轉漬法------------------------------------72
4-3 結果與討論------------------------------------------73
4-3-1細胞內雌激素受體之親和力純化效果 –Blotting detection ----------------------------------------------73
4-3-2細胞內雌激素受體之親和力純化效果 - MS detection-74
4-3-3雌激素金奈米探針對於配位受體之親和力探討 -------75
4-4 結論與未來展望--------------------------------------77

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