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研究生: 林煥雯
Huan-wen Lin
論文名稱: Angiotensin - converting enzyme related, Acer基因在果蠅心臟發育之功能及調控
Function and Regulation of Angiotensin - converting enzyme related gene, Acer, during cardiogenesis of Drosophila
指導教授: 蘇銘燦
Su, Ming-Tsan
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2005
畢業學年度: 93
語文別: 中文
論文頁數: 62
中文關鍵詞: 血管收縮素果蠅心臟發育心臟功能增進子
英文關鍵詞: Angiotensin-converting enzyme related, Drosophila, heart development, tinman, pannier, Hand, enhancer, heart performance
論文種類: 學術論文
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  • Renin-angiotensin system(RAS)的主要功能之一為調控脊椎動物血壓的恆定,Angiotensin-converting enzyme(ACE)家族對於心臟發育及型態有重要的影響,近年研究顯示哺乳類ACE2及其在果蠅的同源基因Angiotensin -converting enzyme related(Acer)在胚胎發育時期表現於於心臟及附屬組織。由於果蠅和脊椎動物在心臟發育的形態和基因調控上有高度的保守性,且為研究心臟發育的優良模式,本研究針對Acer基因的表現與調控Acer做詳細的探討。原位雜合顯示Acer 為母系遺傳基因,最早可在受精卵中偵測到,在胚胎開始發育後隨即消失,直到germ band 完全延伸時表現於造背部的中胚層,當心臟先趨細胞形成後,Acer亦持續地表現在這些心肌細胞,直到外胚層背部癒合,且心肌細胞由兩側移動至背中線處結合形成管狀器官,Acer 在心臟特異性的表現顯示其在心臟發育扮演一定的角色,而先前研究也證實在Acer的突變胚胎心肌及圍心細胞有缺失的性狀。為了解上游調控Acer在心臟功能的基因,我們也找控制Acer在心臟特異性表現的增進子(enhancer) 是位在-188~1211序列間,增進子趨動報導基因的表現型式與Acer相似,研究也証實Tinman (tin)和Pannier (pnr)可直接調控Acer基因的表現,顯示Acer 為tin 及pnr的下游基因。由於脊椎動物ACE也會調控血壓的恆定,近而影響心血管功能,我們也嘗試了解Acer基因的活性是否會影響果蠅心臟功能,初步結果顯示,當Acer過度表現除了少數胚胎心肌細胞增生在胚胎發育時死亡外,大部分的果蠅可存活到成蟲,心臟跳動速率不受影響,但心臟較不能耐受激烈的收縮,顯示Acer在成體果蠅心臟功能上也扮演一定的角色。

    One of the major functions of the Renin-angiotensin system (RAS) is to maintain the homeostasis of blood pressure in vertebrates. Members of the Angiotensin-converting enzyme (ACE) gene family are also involved in heart development and morphogenesis. Previous studies have demonstrated that mammalian ACE2 and its Drosophila counterpart, Acer, are expressed in heart and associated tissues. Since cardiogenesis and regulatory genes that involved in heart development of fly and mammals are evolutionary conserved, and Drosophila as an excellent model system to dissect the genetic factor participated in heart development, we would like to unravel the function of Acer in heart development and dissect up-stream factors that regulate the expression of Acer. The dynamic expression pattern of Acer has been revealed by in situ hybridization. It has been shown that Acer is a maternal gene as it has been detected in fertilized egg. Its maternal expression late is disappeared, and it is expressed in dorsal mesoderm, which late contributes to cardial mesoderm, at germ extension stages. The mesodermally expressed Acer is persisted in heart precursors and persisted till the cardial cells fused to form a cardiac tube underneath the dorsal mid line. As Acer is mainly expressed in heart-specific manner, it suggests that it may function in heart development. Pervious studies have demonstrated that cardial as pericardial cells are missing in Acer mutant embryos. To further understanding the upstream factors that regulate the cardial expression of Acer, we have identified the cardial enhancer of Acer is located between -188 and 1211. The expression pattern of the reporter gene is similar to that of Acer. Further studies had demonstrated that Acer is under the direct control of tinman and pannier. Since ACE is also control the homeostasis of blood pressure, which affects the cardiovascular function of mammals, we would like to learn whether Acer is also function in adult fly. Previously studies suggested that heart beat as well as life span are not affect when Acer is overexpressed. Less than 5% of embryos are dead at late embryogenesis with ectopically cardial cells as Acer activity is elevated. Nevertheless, cardiac performance is great affected when Acer is mis-regulated, suggesting Acer is also function at adult stages.

    壹、摘要 英文摘要------------------------------------ 1 中文摘要------------------------------------ 3 貳、緒論 一、果蠅心臟簡介---------------------------- 4 二、ACE家族對心臟的影響--------------------- 8 三、研究問題與目的-------------------------- 11 參、材料與方法 一、遺傳雜交實驗所使用的果蠅品系------------ 12 二、預測可能是Acer促進子的片段-------------- 12 三、轉殖果蠅的建立-------------------------- 13 四、原位雜合(in situ hybridization)--------- 13 五、反轉錄作用------------------------------ 15 六、冷凍切片-------------------------------- 16 七、檢視果蠅的心臟功能---------------------- 16 肆、結果 一、以原位雜合確定Acer表現的時間與位置------ 17 二、找出調控Acer表現的促進子---------------- 17 三、twi- 24B果蠅株調控的時間和位置---------- 19 四、Tinman對Acer-en1下游基因表現的影響------ 19 五、Tinman對Acer mRNA表現的影響------------- 20 六、Pannier對Acer基因表現的影響------------- 20 七、同時在中胚層過度表現Tinman和Pannier對Acer mRNA表現的影響-------------------------- 21 八、確認BL10679果蠅株中Acer的缺失程度------- 21 九、過度表現Acer對成體果蠅心臟的影響-------- 22 伍、討論 一、Acer在胚胎發育時期專一地表現在心臟------ 24 二、Tinman和Pannier是Acer上游調控因子------- 24 三、建立Acer null mutant-------------------- 26 四、Acer會影響成體果蠅的心臟性能------------ 26 五、Acer是否影響成體果蠅動脈管徑的大小------ 27 六、Basic helix-loop-helix轉錄因子HAND調控心 臟發育---------------------------------- 28 陸、參考文獻-------------------------------- 30 柒、圖表------------------------------------ 35 捌、附錄------------------------------------ 57

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