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研究生:李思怡
研究生(外文):Lee, Sihyi
論文名稱:吳郭魚第三型肝細胞核因子啟動子 片段之功能分析
論文名稱(外文):Functional Analysis of Tilapia (Oreochromis mossambicus) Hepatocyte Nuclear Factor-3β Promoter Fragments
指導教授:黃尉東
指導教授(外文):Huang, Weitung
口試委員:李泰林唐品琦黃尉東
口試委員(外文):Lee, TailinTang, PinchiHuang, Weitung
口試日期:2012-07-24
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
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:109
中文關鍵詞:吳郭魚性腺第三型肝細胞核因子啟動子固醇類荷爾蒙
外文關鍵詞:tilapiagonadhepatocyte nuclear factors-3 (HNF-3)promotersteroid hormones
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第三型肝細胞核因子(hepatocyte nuclear factors-3, HNF-3)家族為富含於肝臟之轉錄因子,其於胚胎發育、分化、代謝及成體時期器官生成扮演重要之角色。本研究室先前於吳郭魚肝臟及性腺中偵測到第一型及第二型類胰島素生長因子(insulin-like growth factors-I/II)基因表現外,亦有HNF-1α、HNF-1β及HNF-3β之基因表現,其中以HNF-3β之表現較其他因子顯著,並可受固醇類荷爾蒙17β-estradiol所調控。因此本研究針對先前所構築之HNF-3β啟動子其0.5 kb、1.0 kb、1.5k及2.0 kb帶有綠螢光報導基因之四片段進行吳郭魚性腺細胞(TO-2)及人類肝癌細胞(Hep3B)轉染並顯微注射斑馬魚受精卵與冷光分析。各片段經顯微注射後24小時觀察發現,於卵黃囊或動物極之體節上有綠螢光之表現,持續觀察96小時後於幼魚之卵黃囊、體節(somites)、脊索(notochord)、底層(floor plate)及眼睛等部位均發現綠螢光之表現。0.5 kb片段之啟動子於脊索、卵黃囊、眼睛及頭部之表現率分別為18.3%、1.3%、35.6%及26.0%;1.0 kb片段之啟動子於脊索、卵黃囊及頭部之表現則分別為約44.4%、44.4%及2.0%;1.5 kb片段之啟動子其表現於脊索及卵黃囊之表現率分別為33.7%與50.5%,而2.0 kb片段之啟動子之表現多於脊索、卵黃囊及頭部,表現率分別為61.2%、26.1%與2.7%,其表現位置大致與先前之研究結果相似。以西方點墨法分析eGFP表現量與冷光分析之結果顯示TO-2及Hep3B細胞經添加17β-estradiol後前二數據均有增加之趨勢。因此推測,位於吳郭魚HNF-3β啟動子之estrogen response element(ERE)結合位置,可經由固醇類荷爾蒙誘導而促進HNF-3β於性腺上表現。




The hepatocyte nuclear factors-3 (HNF-3) family members HNF-3α, HNF-3β and HNF-3γ are hepatocyte-enriched transcription factors, they play important roles in controlling development, differentiation, metabolism and organogenesis. In our previous study, the expression of insulin-like growth factor-I/II (IGF-I/II), HNF-1α, -1β and -3β were detected in the liver and gonads of tilapia, and expreession level of HNF-3β was higher than others and it could be regulated by 17β-estradiol. In this study, four fragments (0.5, 1.0, 1.5 and 2.0 kb) of tilapia HNF-3β promoter were constructed with green fluorescent protein (GFP) gene for biological activity assay by performing transfection into tilapia ovarian cell line (TO-2) and human hepatoma cell line (Hep3B) for western blot analysis and luciferase assay or microinjection into zebrafish eggs and assay. The GFP was mainly expressed in yolk and somites 24 h after injection, in notochord and floor plate 96 h after injection. The 0.5 kb fragment was expressed in notochord, yolk, eye and head, the expression rates were 18.3%, 1.3%, 35.6% and 26.0%, respectively; the 1.0 kb fragment was expressed in notochord, yolk, and head, the expression rates were 44.4%, 44.4% and 2.0%, respectively; the 1.5 kb fragment was expressed in notochord and yolk, the expression rates were 33.7% and 50.5% respectively, and the 2.0 kb fragment was expressed in notochord, yolk and head, the expression rates were 61.2%, 26.1% and 2.7% respectively. The results were similar to our previous studies. The supplement of add 17β-estradiol enhanced western blot analysis of eGFP expression and luciferase assay in TO-2 and Hep3B cells. Based on the present results, hypothesizing that estrogen response element (ERE) in tilapia HNF-3β promoter could promote the expression of HNF-3β in the gonads of tilapia through the action of steroids.


封面內頁
簽名頁
中文摘要........................................................................................ii
英文摘要 ..........................................................................................v
誌謝 .............................................................................................vi
目錄 .............................................................................................vii
圖目錄 ..........................................................................................xi
表目錄 ..........................................................................................xiv
附錄 ..............................................................................................xv

1. 前言 ...........................................................................................1
2. 文獻回顧 .......................................................................................3
2.1 肝細胞核因子(HNFs)之簡介 ......................................................................3
2.2 第三型肝細胞核因子(HNF-3)介紹 ..................................................................4
2.2.1 HNF-3 家族之簡介 .............................................................................4
2.2.2 HNF-3β之結構與生理功能 .......................................................................5
2.2.3 HNF-3與荷爾蒙之相互作用 .......................................................................6
2.3 類胰島素生長因子(insulin-like growth factors, IGFs)與HNF-3β相互間調控及其於性腺表現之相關性 .....6
2.3.1 IGF家族之簡介 ...............................................................................7
2.3.2 IGFs於性腺上發育之作用 ........................................................................7
2.3.3 HNF-3β與IGFs之相關性 ........................................................................8
2.4 研究目的 .....................................................................................9
3. 材料與方法 ...................................................................................11
3.1 試驗材料 ....................................................................................11
3.2 試驗方法 .....................................................................................11
3.2.1 複製選殖載體 ................................................................................11
3.2.1.1 勝任細胞(competent cell)之製備 ...........................................................12
3.2.1.2 轉型作用(transformation)................................................................12
3.2.1.3 小量質體之純化 ............................................................................13
3.2.1.4 大量質體之製備 ............................................................................14
3.2.1.5 限制酶酵素切割反應(restriction enzyme digestion)..........................................15
3.2.2 瓊脂膠體製備與電泳分析 .......................................................................15
2.2.1 1.5℅瓊脂膠體製備 ..........................................................................15
3.2.2.1 瓊脂膠體電泳分析 ...........................................................................15
3.2.3 斑馬魚卵之顯微注射(Microinjection)..........................................................16
3.2.3.1 斑馬魚卵之收集 ............................................................................16
3.2.3.2 顯微注射針之製備(Preparation of injection needles)........................................16
3.2.3.3 顯微注射(Microinjection).................................................................17
3.2.3.4 固醇類賀爾蒙(17β-estradiol)之添加 ........................................................17
3.2.3.5 注射卵之螢光顯微鏡觀 ......................................................................17
3.2.4 細胞株之培養 ...............................................................................18
3.2.4.1 培養細胞株之條件 ..........................................................................18
3.2.4.2 細胞株繼代培養 ............................................................................18
3.2.5 細胞株之轉染(transfection).................................................................19
3.2.5.1 pGFP-1、pEGFP-N1、pGL-3 basic與pGL-3 CMV載體之細胞轉染 .....................................19
3.2.5.2 細胞培養液固醇類賀爾蒙(17β-estradiol)之添加 ...............................................20
3.2.6 西方墨點(Western blot)分析 ................................................................20
3.2.6.1 細胞蛋白質之萃取 ..........................................................................20
3.2.6.2 蛋白質濃度測定 ............................................................................21
3.2.6.3 SDS-PAGE膠體之配製 .......................................................................21
3.6.2.3.1 分離凝膠(separating gel)之配製 .........................................................21
3.6.2.3.2 堆積凝膠(stacking gel)之配製 ...........................................................22
3.6.2.4 SDS膠體電泳分析 ..........................................................................22
3.6.2.5 膠體染色與封膠 ............................................................................23
3.6.2.5.1 Coomassie Brillant Blue R-250 染色法 ..................................................23
3.6.2.5.2 封膠 ..................................................................................23
3.6.2.6 電轉印(electroblotting).................................................................23
3.6.2.7 雜合與偵測(hybridization and detection)..................................................24
3.2.7 影像及軟體分析 ..............................................................................25
3.2.8 冷光分析 ...................................................................................25
3.2.9 半定量分析(semi-quantitation)..............................................................26
3.2.9.1 西方墨點半定量分析 .........................................................................26
3.2.9.2 冷光半定量分析 ............................................................................26
3.3 統計分析 .....................................................................................26
4. 結果 .........................................................................................28
4.1 HNF-3β啟動子之序列 ...........................................................................28
4.2 吳郭魚HNF-3β基因之啟動子功能活性分析 ............................................................28
4.2.1 HNF-3β基因啟動子之顯微注射分析與觀察 ..........................................................28
4.2.2 荷爾蒙誘導HNF-3β啟動子之活性 .................................................................29
4.2.3 pGFP-1及pEGFP-N1載體之細胞轉染與西方墨點法分析 ................................................29
4.2.4 pGL3 basic及pGL3-CMV載體細胞轉染之冷光分析 ...................................................30
4.2.5 荷爾蒙誘導pGFP-1、pEGFP-N1、pGL3 basic與pGL3-CMV四個載體之活性分析 .............................30
5. 討論 ..........................................................................................31
6. 結論 .........................................................................................35
參考文獻 ..........................................................................................83

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