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研究生:林奕岑
論文名稱:tbx5 基因對斑馬魚(Danio rerio)胚胎細胞外基質生成之影響
論文名稱(外文):The impacts of tbx5 gene on extracellular matrix formation during zebrafish (Danio rerio) embryonic development
指導教授:陸振岡
指導教授(外文):Jenn-Kan Lu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:153
中文關鍵詞:斑馬魚細胞外基質
相關次數:
  • 被引用被引用:1
  • 點閱點閱:196
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Tbx5屬於T-box家族中的轉錄因子,在脊椎動物胚胎發育時期表現於心臟、前肢和眼睛。本研究目的為了解tbx5基因對細胞外基質生成的影響。利用專一反意股寡核苷酸抑制斑馬魚胚胎tbx5基因表現,會造成胚胎心臟缺損、心搏減緩、胸鰭發育不全和軀幹彎曲等現象。以斑馬魚DNA晶片偵測tbx5基因弱化後斑馬魚胚胎在24、30及48 hpf基因表現情形,在1.5倍的篩選條件下,表現量上升包括細胞凋亡、抑制細胞增生等功能相關基因。表現量下降包括膠原蛋白、生長激素受體、肌肉調節及發育、細胞生長等功能相關基因。進一步分析顯示,發現細胞外基質相關基因在tbx5基因弱化後表現量下降。利用晶片的結果進行相對即時定量偵測細胞外基質相關基因其表現量都有顯著下降(p<0.05)。免疫螢光染色的實驗結果顯示,經tbx5-MO處理後細胞外基質相關蛋白質表現量皆有下降。利用生長荷爾蒙GH與IGF-Ι改善tbx5基因所造成缺損情形,結果顯示心臟、胸鰭及軀幹的缺損情況減少,心搏數目增加。在斑馬魚DNA晶片偵測tbx5基因弱化後運用生長荷爾蒙GH與IGF-Ι改善缺損的試驗中,以1.5倍的篩選條件下,表現量上升包括細胞增生、肌肉發育及調節、細胞週期等功能相關基因。表現量下降包括細胞增殖及分化、肌肉調節、抑制細胞凋亡及細胞粘連等功能相關基因。在相對即時定量分析結果中細胞外基質相關基因經生長荷爾蒙GH與IGF-Ι處理細胞外基質相關基因表現量皆有上升趨勢。在免疫螢光染色的實驗中,tbx5-MO共同注射生長荷爾蒙GH與IGF-Ι後細胞外基質相關蛋白質表現量皆有上升。本研究結果證明以斑馬魚作為研究細胞外基質形成與心臟發育模式的可能性。將來可應用於治療先天性遺傳疾病與藥物篩選研究之模式。
謝辭 i
摘要 ii
英文摘要 iv
目錄 vi
表目錄 ix
圖目錄 x
壹、前言 - 1 -
一、先天性心臟病(congenital heart disease,CHD) - 1 -
二、胸鰭(pectoral fin) - 6 -
三、軀幹(trunk) - 7 -
四、T-box 轉錄因子(Transcription factor)家族 - 7 -
五、Tbx5基因之表現區域及其作用 - 9 -
六、細胞外基質(extracellular matrix,ECM)對胚胎之影響 - 11 -
七、生長賀爾蒙對胚胎發育之影響 - 22 -
八、應用DNA 微陣列晶片(cDNA microarray)及轉錄體(Transcriptomics)在斑馬魚胚胎發育上之研究 - 25 -
九、本研究之目標 - 27 -
貳、實驗材料 - 31 -
參、實驗方法 - 34 -
一、斑馬魚飼養與受精胚胎之收集及培育 - 34 -
二、斑馬魚sdc4、smad1、smad3a、dag1、colla1、mmp13、tgm2、gh1、mmp14b、fgf1、mmp2、mmp9基因之選殖 - 34 -
三、斑馬魚sdc4、smad1、smad3a、dag1、colla1、mmp13、tgm2、gh1、mmp14b、fgf1、mmp2、mmp9在不同發育時期之相對定量分析 - 41 -
四、顯微注射(Microinjection) - 42 -
五、應用生長相關荷爾蒙growth homone及insulin-like growth factor-Ι改善心臟細胞外基質之研究 - 44 -
六、利用免疫染色觀察tbx5基因弱化後其細胞外基質相關基因表現情形 - 46 -
七、運用斑馬魚晶片(Danio rerio DNA Microarray)探討tbx5基因弱化後心臟相關基因的表現情形 - 48 -
肆、結果 - 52 -
一、利用tbx5專一性反意股寡核酸弱化tbx5基因表現後,觀察斑馬魚胚胎不同發育時期之活存率 - 52 -
二、利用tbx5專一性反意股寡核酸弱化tbx5基因表現後,觀察斑馬魚胚胎各種發育缺損情形的比較 - 52 -
三、利用穿透式電子顯微鏡(TEM)觀察tbx5基因弱化後斑馬魚心臟結構的改變 - 54 -
四、運用斑馬魚晶片 (Danio rerio DNA microarray) 探討tbx5 基因弱化後基因與基因之間的交互作用 - 55 -
五、利用相對即時定量聚合酶連鎖反應偵測tbx5基因弱化後細胞外基質形成相關基因之影響 - 56 -
六、用免疫螢光染色觀察Tbx5基因弱化後斑馬魚心臟及胸鰭組織上細胞基質相關蛋白質之表現 - 58 -
七、觀察tbx5基因弱化後分別注射生長荷爾蒙growth hormone、insulin-like growth factor-Ι,斑馬魚胚胎不同發育時期之活存率 - 58 -
八、觀察tbx5基因弱化後分別注射生長荷爾蒙growth hormone、insulin-like growth factor-Ι,斑馬魚胚胎各種發育缺損情形的比較 - 59 -
九、運用斑馬魚晶片 (Danio rerio DNA microarray) 探討tbx5 基因弱化後運用生長荷爾蒙growth hormone、insulin-like growth factor-Ι改善其缺損的基因交互作用 - 63 -
十、利用相對即時定量聚合酶連鎖反應偵測tbx5基因弱化後運用生長荷爾蒙growth hormone、insulin-like growth factor-Ι改善其細胞外基質形成相關基因之影響 - 65 -
十一、用免疫螢光染色觀察Tbx5基因弱化後運用生長荷爾蒙growth hormone、insulin-like growth factor-Ι改善其斑馬魚心臟及胸鰭組織上細胞基質相關蛋白質之表現 - 68 -
伍、討論 - 70 -
一、利用tbx5專一性反意股寡核苷酸弱化tbx5基因表現後,觀察斑馬魚胚胎不同發育時期之活存率 - 70 -
二、利用tbx5專一性反意股寡核苷酸弱化tbx5基因表現後,觀察斑馬魚胚胎各種發育缺損情形的比較 - 70 -
三、利用穿透式電子顯微鏡(TEM)觀察tbx5基因弱化後斑馬魚心臟結構的改變 - 71 -
四、運用斑馬魚晶片 (Danio rerio DNA microarray) 探討tbx5 基因弱化後基因與基因之間的交互作用 - 72 -
五、利用相對即時定量聚合酶連鎖反應偵測tbx5基因弱化後細胞外基質形成相關基因之影響 - 74 -
六、用免疫螢光染色觀察tbx5基因弱化後斑馬魚心臟及胸鰭組織上細胞基質相關蛋白質之表現 - 75 -
七、觀察tbx5基因弱化後分別注射生長荷爾蒙growth hormone、insulin-like growth factor-Ι,斑馬魚胚胎不同發育時期之活存率 - 75 -
八、觀察tbx5基因弱化後分別注射生長荷爾蒙growth hormone、insulin-like growth factor-Ι,斑馬魚胚胎各種發育缺損情形的比較 - 76 -
九、運用斑馬魚晶片 (Danio rerio DNA microarray) 探討tbx5 基因弱化後運用生長荷爾蒙growth hormone、insulin-like growth factor-Ι改善其缺損的基因交互作用 - 77 -
十、利用相對即時定量聚合酶連鎖反應偵測tbx5基因弱化後運用生長荷爾蒙growth hormone、insulin-like growth factor-Ι改善其細胞外基質形成相關基因之影響 - 78 -
十一、用免疫螢光染色觀察tbx5基因弱化後運用生長荷爾蒙growth hormone、insulin-like growth factor-Ι改善其斑馬魚心臟及胸鰭組織上細胞基質相關蛋白質之表現 - 79 -
陸、參考文獻 - 81 -
柒、圖表 - 108 -
捌、附錄 - 150 -


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