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研究生:謝丞智
研究生(外文):Hsieh, Cheng-Chih
論文名稱:研究斑馬魚過量表現活化轉錄因子4誘導脂肪細胞分化之功能
論文名稱(外文):The Functional Study of Activator Transcription Factor 4 (ATF4) Leads to Adipose Differentiation in Zebrafish
指導教授:何國牟
指導教授(外文):Her, Guor-Mour
口試委員:許準榕劉秉慧葉光揚
口試委員(外文):Sheu, Joen-RongLiu, Biing-HuiYeh, Kun-Yun
口試日期:2016-07-20
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生命科學暨生物科技學系
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:2016
畢業學年度:104
語文別:中文
論文頁數:47
中文關鍵詞:ATF4脂肪細胞分化
外文關鍵詞:ATF4adipocyte differentiation
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ATF4是一種活化轉錄因子,它屬於cyclic adenosine monophosphate responsive element
-binding(CREB) 蛋白家族的一員,當細胞內有內質網壓力產生,就會使ATF4的表現量上升,進而促進下游基因的轉錄,例如CHOP、Atg12、E-selection,進行未摺疊蛋白質反應(UPR),幫助細胞度過內質網壓力。在過去有文獻指出,在小鼠的胚胎纖維母細胞過量表現ATF4,會提高脂肪的生成,並且用siRNA將ATF4進行抑制的情況下,會阻斷脂肪前驅細胞轉換成脂肪細胞,且許多脂肪細胞分化相關的基因例如C/EBPβ、PPARγ、C/EBPα等等,表現量都明顯的下降。在過去的文獻也有指出ATF4直接調控許多與脂肪分化或是代謝的相關基因例如APOE、Ihh、C/EBPβ、PPARγ、FGF19、ASNS。斑馬魚胚胎發育的透明性為探討脂肪細胞生物學機制提供了新的機運,我們可以透過螢光顯微鏡觀察斑馬魚的胚胎是否有過量表現我們所要研究的基因,因此在本篇研究中希望能建立一個大量表現ATF4基因的斑馬魚模式生物,我們將ATF4的基因轉殖入構築好的轉基因載體(Vector),再透過顯微注射 (Microinjection) 的方式注射到斑馬魚的胚胎(embryo)中,透過全身表現的Beta-actin啟動子(B-act),可以在全身B-actin表現外源性的ATF4並帶有紅螢光蛋白,搭配tet-off系統,添加藥物Doxycycline能夠任意的關閉基因,便能夠觀察及研究斑馬魚早期的脂肪細胞分化。

ATF4 (activator transcription factor4) is a activator transcription factor. It is a member of cyclic adenosine monophosphate responsive element-binding protein family. When endoplasmic reticulum stress exerts an influence to cells, the expression of ATF4 will be upregulated in the cytoplasm. Then the transcription of ATF4 downstream genes such as CHOP (CCAAT-enhancer-binding protein homologous protein), Atg12 (Autophagy-related protein 12), E-selection are promoted. The unfolded protein response is carried out after their transcription are promoted and supports cells to tide over endoplasmic reticulum stress. Previous several studies demonstrated that overexpression of ATF4 in the embryo fibroblast of the mouse increased adipocyte differentiation. In addition to, silenced ATF4 by siRNA blocked conversion of preadipocytes to adipocytes and a lot of expression of genes associated with adipocyte differentiation such as PPARγ (peroxisome proliferator-activated receptor gamma), C/ EBPβ (CCAT/enhancer-binding protein beta), C/EBPα (CCAT/ enhancer- binding protein alpha), SCD1 (stearoyl CoA desaturase1) were significantly downregulated. Previous studied indicated that ATF4 directly targeted many genes related to adipogenesis and lipid metabolism such as C/EBPβ, PPARγ, FGF19 (Fibroblast growth factor 19), RUNX2a (Runt-related transcription factor 2). The transparent embryos of the zebrafish provide new selection to research adipose biology. We could observe embryos whether they are overexpressed ATF4 with fluorescence microscopy. Therefore, we expect to set up a ATF4 overexpression transgene zebrafish model in our study. We transfected ATF4 gene into constructed transgene vector included tet-off system and microinject this construct into embryos. They would express whole body transgene by β-actin promoter. The whole body β-actin expresses exogenous ATF4 and carry red fluorescent protein. We could turn off expression of ATF4 in this model fish by adding doxycycline. Continuously, we used oil red O staining to assay adipose differentiation and lipid accumulation and detect the expression of
ATF4 targeted genes in embryos in7dpf(day-post fertilization) and 21dpf. It provided a platform to study adipogensis in zebrafish early stage.

謝辭………………………………………………………………………………………...I
Abstract……………….………………………………………………………………………II
摘要………………………………………………………………………………………III
目錄………………………………………………………………………………………IV
圖目錄…………………………………………………………………………………….V
表目錄……………………………………………………………………………………VI
壹、序論…………………………………………………………………………………..1
(一) 動物模式-斑馬魚………….……………………………………………………..1
(二) beta-actin promoter 簡.………………………………….……..1
(三) Tol2 systems簡介……………………………………………………………...2
(四) Tet-off systems簡介……………………………………………………...2
(五) ATF4……………………………..…..……………...………………………………….2
(六) Oil Red-O lipid stain…………………………………………………..3
(七)H&E染色…………………………..………………………………………………3
(八)脂肪細胞分化…………………………………………………………………………3
(九)研究動機………………………………………………….……………………….4
貳、材料與方法………………….…………………………………………………………..5
(一)材料………………….…………………………………………………………….5
一、生物材料……………………………………………………………………..5
二、反應試劑……………………………………………………………………..5
三、儀器…………………….……………………………………………………..8
(二)實驗方法………………………………………………………………………….8
一、生物技術…………………………………………………………………………8
二、β-actin ATF4 Construct 構築…………………………………………….17
三、基因轉殖斑馬魚之建立………………….…………………………………….18
參、實驗結果……………………………………………………………………………….21
(一) β-actin-tet-off-ATF4轉基因斑馬魚模式建立………….………………………21
(二) Doxycycline關閉外源性ATF4基因……………………..………………………..21
(三) Oil Red O染色…………………..…………………………..……………………….21
(四) 高油脂餵食下,ATF4標的基因及脂肪細胞合成基因半定量………………22
(五) β-actin-tet-off-ATF4轉基因於組織切片…………………………….23
肆、討論………………………………………………………………………..………….24
伍、圖……………………………………………………………..………………………….27
陸、表……………………………………………………………..……………………….44
柒、參考文獻……………………………………………………………………………..…45


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