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研究生:李靜怡
研究生(外文):Jing-Yi Li
論文名稱:Drosophilaangiotensinconvertingenzyme-related(Acer)基因於果蠅心臟發育之功能探討
論文名稱(外文):Function of Angiotensin converting enzyme -related gene in Heart development of Drosophila melanogaster
指導教授:蘇銘燦
指導教授(外文):Ming-Tsan Su
學位類別:碩士
校院名稱:國立臺灣師範大學
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:65
中文關鍵詞:心臟發育果蠅
外文關鍵詞:heart developmentcardiogenesisDrosophilaacerACEangiotensin converting enzymeangiotensin converting enzyme-related
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果蠅心臟〈背血管〉由104個可分為數群表現不同分子標誌及功能的細胞組成,由於其結構相較簡單因此為一研究心臟發育很好的模式動物,本研究嘗試利用此一模式探討Acer (Angiotensin Converting enzyme- related) 在果蠅心臟發育中的功能。Acer為一個具金屬性胜肽酶活性區〈Metallopeptidase active site〉的酵素,先前報告顯示其在果蠅心臟發育有一定的功能,但其角色為何並未詳加研究。原位雜合實驗証實晚期Acer表現於果蠅胚胎的背血管中。以心臟細胞分子標誌 (如:Tinman、eve和Odd等) 進行抗體染色則顯示,在Acer突變株中顯現心肌及圍心細胞缺失及分布異常的性狀;相反地,過度表現Acer則發現會導致心肌及圍心細胞增多的現象,這樣的性狀與Tin、Pnr非常類似,顯示在心臟發育的過程中Tin、Pnr及Acer 極可能作用於同一路徑,初步研究証實Acer可能與Pnr有交互作用,Acer 是否與tinman 及pnr 交互作用或是單獨作用於果蠅心臟發育,則須更多研究証明。
Heart of Drosophila consists of 104 cells which are classified into different groups according the specific cell markers and functions. As its structure is relative simple, this makes Drosophila is one of the most excellent models to study the cardiogenesis of animals. In this study we plant to study the function of acer in heart development using Drosophila as a model system. Acer is a metallopeptidase. Previous studies suggested that it may function in development heart. Nevertheless, its role in heart morphogenesis has not been characterized in detail. in situ hybridization demonstrates that acer is expressed exclusively in heart of Drosophila (dorsal vessel). Immunocytochemistry stainings using different heart-specific markers, such as tinman, odd and eve, have revealed that cardial and pericardial cells are missing or misplaced in acer mutant embryos. By contrast, overexpression of Acer results in overproduction of cardial and pericardial cells. As the heart phenotypes of acer were similar to that of tinman and pnr, this indicate that acer, tinman and pnr act in the same pathway during heart morphorgenesis in flies. Preliminary studies demonstrated that acer may interact with tin and pnr. Further studies are needed to demonstrate if acer acts in concert with tin and pnr or function along to specify different cardial cell types.
壹、英文摘要………………………………………...………………………5
中文摘要………………………………………………………………6
貳、緒論……………………………………………………………...……....7
一、研究背景 ……………………………………………………………7
(一) 果蠅之心臟 .……………………………………………………7
(二) 果蠅之心臟發育與其基因調控 .…………………………...….7
(三) 哺乳動物之ACEs ..……………………………………………11
(四) 果蠅中ACE的同源基因Acer (Angiotensin converting enzyme-related)………………………………………………...13
二、研究目的 ……………………………………...………………….17
參、材料與方法 ………………………………………………………...…19
一、 研究材料:果蠅的取得及維持 ………………………..…..19
二、 研究方法 ………………………………………………..…19
(一) Clone Acer cDNA to Vector pUAST ..……………………....…19
1. Midi-preparation of Acer cDNA plasmid .……………...……19
2. Enzyme digestion and ligation .………………………………20
3. Transformation以及轉殖菌株之篩選 ………………….…21
(二) In Situ Hybridization ..…………………………………………22
1. prepare RNA probe .……………………………………….....22
2. 收集並固定胚胎 …………………..…………………..……22
3. hybridization .…………………………………………...……23
(三) 果蠅胚胎之抗體染色.…………………………………………23
1. 收集並固定胚胎 ………………………………………....…23
2. 抗體染色 ……………………………………………..…..…24
肆、結果 ……………………………………………………………...……25
一、 Acer基因表現於胚胎發育晚期之背血管中……………….….…25
二、 Acer基因功能缺失(lose-of-function)對晚期之心臟發育有顯著影響。.………………………………………………………...……25
三、 Acer基因功能獲得(gain-of-function)造成心臟型態的改變。…..30
四、 與Tin、Pnr間的交互作用…………………………………………33
伍、討論 ………………………………………………………………...…35
一、 acer基因的表現對果蠅心臟的正常發育〈包括心細胞(Cardiac Cell)以及圍心細胞(Pericadiac cell)〉是必須的。.……………...…35
二、 acer基因的重要功能之一可能與圍心細胞在背部癒合過程中的移動有關。 …………………………………………………..……35
三、 acer基因需要中胚層的內含物來完成其正常功能。 ………..…36
四、 acer基因的表現不直接受到tin、pnr的調控;而pnr與acer基因的調控有關。 …………………………………………………..…37
陸、參考文獻 .………………………………………………….…….……55
圖目錄
圖一、以野生型〈Wild Type:W1118〉果蠅胚胎之原位雜合(In Situ Hybridization)………………………………………………..……39
圖二、Acer基因功能缺失〈lose-of-function〉對發育晚期表現Tin心臟細胞之數目以及細胞分布有顯著影響……………………..……40
圖三、Acer基因功能缺失〈lose-of-function〉對發育晚期表現Eve之圍心細胞之數目有顯著影響…………………………………..……42
圖四、Acer基因功能缺失〈lose-of-function〉使果蠅心臟中表現Odd之圍心細胞數目減少且有細胞分布位置上的異常…………..…44
圖五、Acer基因功能缺失〈lose-of-function〉的果蠅胚胎………………46
圖六、確認UAS-gal4的系統,的確能將Acer過度表現於所需要研究的時期及位置……………………………………………………..48
圖七、在外胚層過度表現Acer於不會影響果蠅心臟表現Tin之細胞...49
圖八、於中胚層過度表現Acer使果蠅胚胎心臟表現Tin的圍心細胞產生分布上的型態異常…………………………………………..…50
圖九、於中胚層過度表現Acer使果蠅胚胎心臟表現Odd的圍心細胞產生嚴重的型態異常,細胞無法正常的移向背中線………………52
圖十、Tinman的缺失〈lost of function〉以及過度表現〈gain of function〉未明顯影響Acer mRNA的表現………………………………….53
附圖一 …………………………………………………………………...Ⅰ
附圖二 …………………………………………………………………...Ⅱ
附圖三 …………………………………………………………………...Ⅲ
附圖四 …………………………………………………………………….Ⅳ
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