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研究生:李彥芸
研究生(外文):Yen-Yun Lee
論文名稱:人體細胞內雌激素受體傳訊系統與螢光報導基因之建構
論文名稱(外文):Development of Estrogen Receptor-Mediated GFP Reporter Gene System in Human Cell
指導教授:楊秋英
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:62
中文關鍵詞:雌激素受體反應基因螢光報導基因雌激素作用
外文關鍵詞:estrogen responsive elements (EREs)green fluorescence protein (GFP)estrogenic
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本研究乃建立人體細胞內雌激素受體反應基因 (estrogen responsive elements, EREs) 與螢光報導基因 (green fluorescence protein, GFP) 的檢測系統,以作為測試具雌激素作用之化合物用。第一階段利用TR-PCR技術倍增n個ERE基因複本的DNA片段 (nERE),接合到pGEM-T Easy Vector後,再轉送 (transformation) E. coli宿主細胞內抽取其質體,以EcoR I/BamH I限制酶切割與定序,得到pGEM-3,4,6.5,7ERE等四株重組質體。第二階段建構帶有螢光報導基因之重組質體,將pGEM-3,4,6.5,7ERE以EcoR I/BamH I限制酶切下3、4、6.5及7個ERE基因複本的DNA片段後,接合到pEGFP-1之EcoR I/BamH I切位上,再轉送E. coli宿主細胞內抽取其質體,以EcoR I/BamH I限制酶切割,得到pEGFP-3,4,6.5,7ERE四株重組質體。第三階段為建構表現質體,所以參考pRL-TK vector內之TK promoter序列,設計TK-F與TK-R引子進行PCR反應增幅TK promoter片段後,嵌入到pEGFP-3,4,6.5,7ERE四株質體之BamH I/Age I切位上,選殖得到pEGFP-3,4,6.5,7ERETK四株重組表現質體。第四階段將pEGFP-3,4,6.5,7ERETK四株表現載體轉染於MCF-7細胞株,篩選出MCF-3ERETK、MCF-4ERETK、MCF-6.5ERETK及MCF-7ERETK細胞株。最後階段為檢測此報導基因系統之可運作性,將此四種細胞株經10 nM E2 (agonist, 促進劑) 處理後皆能活化螢光表現;1 μM ICI (antagonist, 拮抗劑) + 10 nM E2處理後則可抑制螢光之表現,且隨著ERE片段越多其產生之螢光量越高。MCF-3ERETK與MCF-7ERETK細胞株加入具雌激素作用之BPA與NP反應後,從0.01~10 μM可誘發螢光產生,所以未來將檢測更多具雌激素作用之化合物,加以證明系統的可運作性。
This study is trying to set up an estrogen receptor-mediated GFP reporter gene system which can be used to detect estrogenic compounds abounded in environment. First of all, estrogen responsive elements (EREs) fragments were amplified with TR-PCR method and inserted into pGEM-T Easy vector. These resulting products, including pGEM-3ERE, pGEM-4ERE, pGEM-6.5ERE, and pGEM-7ERE, were named by their total numbers of EREs. Second, pGEM-3, 4, 6.5, 7 ERE were digested with EcoR I/BamH I, and then 3, 4, 6.5, and 7 ERE-copies products were cloned into pEGFP-1 vector. Third, thymidine kinase (TK) promoter fragments were inserted into pEGFP-3, 4, 6.5, 7ERE plasmids. Forth, the expression plasmids pEGFP-3, 4, 6.5, 7ERETK were transfected into human breast cancer cell line, MCF-7, and these final cell lines were named MCF-3ERETK, MCF-4ERETK, MCF-6.5ERETK, and MCF-7ERETK. Finally, estrodial (estrogen agonist) and ICI (estrogen antagonist) were introduced to validate these assay systems. As a result, we demonstrate that multiple EREs can increase transcriptional activity. In addition, well-known estrogenic chemicals, BPA and NP, were also introduced and resulted in significant EGFP expression ranging from 0.01 to 10 μM in MCF-3ERETK and MCF-7ERETK cell lines. In the future, MCF-3, 4, 6.5, 7 ERETK cell lines could be used for screening potential environmental estrogenic compounds around environment.
中文摘要……………………………………………………i
英文摘要……………………………………………………ii
目次……………………………………………………iii
表次……………………………………………………………………v
圖次…………………………………………………………………vi
前言……………………………………………………………………1
一、環境賀爾蒙所引發之問題…………………2
二、環境賀爾蒙之離體外生物檢測方法………………………3
三、報導基因種類………………………………5
四、雌激素受體類型……………………………………………………5
五、多重複雌激素受體反應基因之表現……………………7
六、研究目的……………………………………………………………7
材料與方法……………………………………………………………9
一、試驗材料………………………………………………………9
(一) 試驗藥品……………………………………………………9
(二) 試驗引子、DNA 定序與相關之溶液配方………………………9
二、試驗方法……………………………………………………10
(一) 雌激素受體反應基因之備製………………………10
(二) 重組DNA 技術………………………………………………10
(三) 重組質體pGME-nERE 之構築…………………………………12
(四) 重組質體pEGFP-nERE 之構築………………………………………13
(五) 表現質體之構築……………………………………………13
(六) 蛋白抽取與西方墨點法……………………………………14
(七) MCF-7 細胞培養與轉染作用………………………16
(八) 螢冷光儀分析螢光量之表現………………………………17
(九) 統計分析……………………………………………………17
結果……………………………………………………18
一、建構以雌激素反應元素調控GFP 報導基因之系統…………………… 18
(一) 以TR-PCR 及Adapter-PCR 反應產生nERE………………………… 18
(二) 四株重組質體pGEM-3,4,65,7ERE 之分析…………………………18
(三) 四株重組質體pEGFP-3,4,65,7ERE 之分析…………………………19
(四) 表現載體之分析………………………………………………………19
二、以西方墨點法探測帶有ERα與ERβ之細胞株………………………20
三、四種不同啟動子之自體螢光表現分析…………………………………21
四、雌激素受體傳訊系統與螢光報導基因之分析…………………………21
(一) 四種細胞株MCF-3,4,65,7ERETATA 之螢光表現………21
(二) 四種細胞株MCF-3,4,65,7ERETK 經E2 與ICI 藥劑處理後之不同時間點的螢光表現情形…………………………21
(三) 四種細胞株MCF-3,4,65,7ERETK 與MCF-TK 細胞株之不同重複ERE 片段的螢光表現…………………………………………………22
(四) MCF-7ERETK 細胞株經不同濃度之E2 的螢光表現………………22
(五) 檢測具estrogen agonist 之BPA 與NP 的分析………………………22
討論………………………………………………………………………………24
參考文獻…………………………………………………………………………51
附錄………………………………………………………………………………57
表次
表一、本試驗所使用之引子……………………………………………………28
表二、本試驗所使用之Escherichia coli 品種簡介……………………………29
表三、比較各檢測系統間誘發E2 之最大反應濃度………………………29
圖次
圖一、TR-PCR 技術倍增n 個EREs 基因複本的DNA 片段流程圖…30
圖二、重組質體pGEM-nERE 之構築圖……………………31
圖三、重組質體pEGFP-nERE 之構築圖………………………………32
圖四、重組表現質體pEGFP-nERETK 之構築圖………………………………33
圖五、洋菜膠體電泳分析TR-PCR 與Adapter-PCR 之合成產物……………34
圖六、洋菜膠體電泳分析重組質體pGEM-nERE………………………………35
圖七、四株重組質體 pGEM-3ERE、pGEM-4ERE、pGEM-65ERE及pGEM-7ERE 之核酸定序………………………………36
圖八、洋菜膠體電泳分析四株重組質體pEGFP-3,4,65,7ERE………………37
圖九、四株重組質體pEGFP-3ERE、pEGFP-4ERE、pEGFP-65ERE 及pEGFP-7ERE之核酸定序………………………………38
圖十、洋菜膠體電泳分析四株重組表現質體pEGFP-3,4,65,7ERETK………39
圖十一、四株重組表現質體pEGFP-3ERETK、pEGFP-4ERETK、pEGFP-65ERETK 及pEGFP-7ERETK 之核酸定序………………40
圖十二、洋菜膠體電泳分析三株重組表現質體pEGFP-3ERECMV、pEGFP-3ERESV40 與pEGFP-3ERETATA……………………………41
圖十三、三株重組表現質體pEGFP-3ERECMV、pEGFP-3ERESV40 及pEGFP-3ERETATA 之核酸定序………………………………42
圖十四、西方墨點法分析12 種細胞株之ER α 與ER β 蛋白質表現……………43
圖十五、比較四種起動子之自體螢光表現…………………………………44
圖十六、螢冷光儀分析MCF-3ERETATA、MCF-4ERETATA、MCF-65ERETATA與MCF-7ERETATA 細胞株之螢光表現………………45
圖十七、螢冷光儀分析MCF-3ERETK、MCF-ERETK、MCF-65ERETK 與MCF-7ERETK 細胞株不同時間點之螢光表現……………………46
圖十八、螢冷光儀分析不同重複nEREs 片段的螢光表現…………………47
圖十九、以E2 藥劑處理MCF-7ERETK 細胞株之螢光表現…………………48
圖二十、以BPA 與NP 藥劑處理MCF-7ERETK 與MCF-3ERETK 細胞株之螢光表現……………………………………………49
圖二十一、螢光顯微鏡觀察經不同藥劑處理MCF-7ERETK 細胞株螢光表現……………50
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