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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/16938

Title: 應用生長相關激素改善tbx5弱化所造成的心臟缺損及以cDNA晶片探討tbx5弱化後斑馬魚胚胎心臟相關基因的表現模式
Development of a heart defect rescue protocol and apply DNA microarray to determine genes expression profile on tbx5 knockdown Zebrafish (Danio rerio)
Authors: Jau-Fen Shiue
薛招芬
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 心臟發育;生長相關荷爾蒙;tbx5
heart development;growth related hormone;tbx5
Date: 2008
Issue Date: 2011-06-30T08:45:06Z
Abstract: Tbx5屬於T-box家族之轉錄因子,表現於脊椎動物胚胎時期的心臟、前肢和眼睛。先前研究結果表明以morpholino antisense RNA抑制斑馬胚胎tbx5基因表現,除了會造成胚胎心臟缺損,也會造成心臟肌肉生成基因及調控胚胎心臟發育之轉錄因子的表現量下降。本實驗的目的是以斑馬魚作為模式魚種,用以促進心肌生長之生長相關激素(hGH、IGF-I及 Ghrelin)處理tbx5基因弱化的斑馬魚胚胎探討改善心臟缺損的情形及利用cDNA microarray研究tbx5弱化後影響斑馬魚心臟發育相關基因的表現模式。實驗結果顯示,在1-4細胞時期處理生長相關荷爾蒙可促使心臟肌肉生成基因-cmlc2、amhc、vmhc及早期胚胎心臟發育轉錄因子-tbx5, dHAND, nkx2.5, myoD, myostatin的表現量上升,以全覆式定位雜交可觀察到分別以GH、Ghrelin及IGF-I 處理後心臟肌肉生成基因cmlc2, amhc, vmhc及tbx5的表現量亦有改善。由於tbx5弱化後會導致胚胎心臟型態的改變,更進一步的由細胞層面探討,結果發現,細胞週期相關基因例如p27, p57, cdk2及PCNA的表現量會增加,可能會導致細胞週期的延長分析細胞凋亡相關基因例如Bax, Bad基因表現量上升,推測此一影響可能導致細胞凋亡加速進行。雖然抗細胞凋亡基因Bcl2其表現量有上升的趨勢,卻可延遲細胞週期G0/S,導致細胞週期的延長,由上述結果得知tbx5基因弱化後會造成心肌細胞數目減少,導致心臟型態改變及功能不全。以斑馬魚cDNA晶片探討tbx5基因弱化後心臟相關基因的表現情形與相互關係。結果顯示,在心管形成24 hpf及心臟looping 36 hpf,tbx5基因主要可下調控胚胎心臟發育相關基因包括fgf8, tbx20 、心肌生成相關基因包括myotrophin, catenin、肌肉收縮相關基因包括troponin T2 cardiac等基因表現量下降,此外,還會啟動細胞凋亡相關基因tumor protein p53 , Fas associated factor 1等基因,推測tbx5弱化會導致心臟心管形成及looping的缺損,進而使心臟發育不完全。
Tbx5 is a member of the T-box family of transcription factors which expresses in the heart, fore limbs/pectoral fins and eyes of vertebrate animals during embryonic development. Previous studies indicated that growth related hormones play important roles throughout heart development and cardiomyogenesis. The goal of present study is to elucidate the impacts of using growth-related hormone-GH, Ghrelin and IGF-I rescue heart defect protocol on the expression of cardiomyogenesis and heart development regulatory factors, and using cDNA microarray to analyze heart development related genes network in the impacts of tbx5 knockdown embryos. The results of present study demonstrated that using growth related hormone can increase cardiac myogenesis genes amhc, vmhc, cmlc2 and transcription factors tbx5, dHAND, nkx2.5, myoD, myostatin decreased significantly. By the knockdown of tbx5 gene. The results indicated that growth related hormone can rescue heart defect in tbx5 knockdown zebrafish embryos. It suggested that the knockdown of tbx5 cause heart defect can be restore heart development and its function by using growth related hormone administration. The tbx5 knockdown the heart defect caused by tbx5 gene knockdown is as a consequence of arresting G1/S-phase of cell cycle,as demonstrated by a dramatic increase in the expression of genes associated with the cardiac cell cycle,including cdk2, PCNA, p27, p57. Tbx5 knockdown also leaded to a increasing of apoptosis-related genes expression increase,including bax, bad and antiapoptosis gene bcl2. Bcl2 was demonstrated could delay G0 to S transition. It suggests tbx5 gene knockdown can delay or arrest at cell cycle G1/S-phase and trigger cell apoptosis coincided with a decreased in the embryonic cell numbers result in heart defect ultimately. A gene experiment cDNA microarray analysis was performed to identify tbx5 knockdown profile and interaction involved in heart development. The results showed that related genes such as the expression of early cardiogenesis , fgf8, tbx20 cardiomyogenesis related genes such as myotrophin, catenin and muscle contraction related genes, such as, troponin T2 cardiac were down-regulated following tbx5 gene knockdown. On the other hand apoptosis related genes such as tumor protein p53 , Fas associated factor 1 was up-regulated following tbx5 knockdown at 24, 36 hpf stages.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M95330017
http://ntour.ntou.edu.tw/ir/handle/987654321/16938
Appears in Collections:[水產養殖學系] 博碩士論文

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