臺灣博碩士論文加值系統

摘要
p53是一種腫瘤抑制蛋白，能對發生DNA損害的細胞作用包括停止細胞週期、細胞凋亡以及細胞衰老。Mdm2是一種致癌基因，具有E3泛素連接酶的功能，會對p53行泛肽化作用，使p53-泛素複合體被26s蛋白質分解酶所辨認將其分解。Gankyrin也屬於一種致癌基因，通常在肝癌細胞中常發現，並可以增加MDM2對p53行泛肽化作用的效率，導致p53表現量的下降，停止細胞凋亡作用。本實驗我們會利用肝臟脂肪酸結合蛋白(L-FABP)的啟動子來建構一個肝臟專一性表現的系統，並分別銜接上GFP-mdm2與GFP-Gankyrin的報導基因與致癌基因，來觀察MDM2與Gankyrin在斑馬魚肝臟中的影響，首先使用顯微注射技術將已建立好的構築體注射入野生種斑馬魚胚體中進行觀察，並分析利用基因體DNA聚合酶連鎖反應及綠螢光蛋白的表現，篩選出成功轉殖的第一代斑馬魚(F0)，將其與野生種交配繁延次代(F1；heterozygote)，可發現在3~5天的轉殖基因斑馬魚F1中，透過螢光顯微鏡可以明顯觀察到肝臟有專一性綠螢光蛋白的表現，此代表mdm2 /Gankyrin的基因成功轉殖到斑馬魚染色體內並可以遺傳到子代，隨後在利用西方點末雜交分析法與逆轉錄聚合酶連鎖反應，篩選出F1中MDM2/Gankyrin表現量最強的轉殖基因斑馬魚，使其自交生出F2；homozygote。未來可利用西方點末雜交方式，來觀察MDM2/
Gankyrin種魚對p53蛋白表現量的影響，及MDM2與Gankyrin種魚兩者所交配的轉殖基因魚中p53被降解的程度。所以在建立肝臟專一性且具有致癌基因的轉殖基因系統後，可以探討相關早期肝臟基因的表現，以及觀察胚胎時期斑馬魚的肝臟發育，有利於後續抗癌藥物的建立與篩選。

Abstract
The p53 is a tumour suppressor protein.The ability of p53 to suppress tumorigenesis is mediated through its response to cellular stress, which culminates in cell growth arrest, apoptosis or senescence.The mdm2 (murine double minute 2 ) gene is an oncogene. In unstressed cells, p53 is maintained at low levels by the action of MDM2, an oncogenic E3 ubiquitin ligase (ubiquitin-protein ligating enzyme).The E3 ubiquitin ligase activity of MDM2 is essential to mediates p53 ubiquitination and proteasomal degradation.Gankyrin is an liver oncoprotein over-expressed in most hepatocellular carci-nomas (HCCs). Gankyrin can activate the ubiquitin protein ligase MDM2 which can ubiquitinate p53 leading to the proteasomal degradation of p53.We used liver fatty binding protein (L-FABP) promoter to establish liver-specific system expression in zebrafish. Then，combine GFP(Green Fluorescence protein).In this experiment, we use L-FABP(liver fatty acid binding protein) promoter to build a represent-specificity of liver system which co-operate GFP(green fluorescence protein) and MDM2/Gankyrin to observe the represent of MDM2 and Gankyrin in liver. We microinjected fusion DNA to wild type embryo, and use genomic DNA PCR and GFP expression to get the transgenic line（F0）. The transgenic line would propagate heterozygote（F1）, and we observed there are specificity of liver in green fluorescence protein by Fluorescence Microscopy in 3-5 days fish. It presents MDM2/Gankyrin was succeeded transgenic into zebrafish and would pass to the next generation. Then we used western blotting assay and RT-PCR to sieve the most strong expression in MDM2/Gankyrin transgenic fish, and crossed them to have the homozygote（F2）. Observing the decrease of P53 of MDM2/Gankyrin in F2 and the level of P53 denatured. So we built a system that has specificity of liver and transgenic line in oncogene, we could understand the specific gene expression. Then we could observe the early stage of zebrafish embryo, making it use for building and selecting anticancer drag.

目錄
中文摘要
英文摘要
壹、序論………………………………………………………………………………01
(一) 動物模式物種-斑馬魚……………………………………………………01
(二) 斑馬魚肝臟發育…………………………………………………………02
(三) L-FABP啟動子簡介………………………………………………………04
(四) p53簡介…………………………………………………………………05
(五) MDM2簡介………………………………………………………………10
(六) Gankyrin簡介……………………………………………………………13
(七) 泛素-蛋白質分解酶途徑………………………………………………15
(八) 研究動機…………………………………………………………………17
貳、材料與方法……………………………………………………………………19
(一) 材料…………………………………………………………………………19
A. 生物材料……………………………………………………………19
B. 儀器…………………………………………………………………19
C. 反應試劑……………………………………………………………20
(二) 實驗方法……………………………………………………………………24
A. 表現載體之構築……………………………………………………25
B. 基因轉殖魚之建立…………………………………………………31
C. Mdm2與Gankyrin基因轉植株的建立與表現…………………33
參、實驗結果………………………………………………………………………40
(一) 利用核酸引子確認構築體的表現………………………………………40
(二) 螢光顯微鏡觀察斑馬魚的表現型………………………………………40
(三) F0-Genomic DNA分析…………………………………………………41
(四) 螢光顯微鏡觀察F1表現型………………………………………………42
(五) F1反轉錄聚合酶鏈鎖反應分析…………………………………………42
(六) F1成魚與F2幼魚之西方墨點法分析……………………………………43
(七) F2利用冷凍切片觀察肝臟發育…………………………………………45
(八) 觀察成魚LF2.8-GFP-mdm2-V5的表現型……………………………46
肆、討論……………………………………………………………………………48
伍、參考文獻………………………………………………………………………57
陸、實驗附圖………………………………………………………………………61

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