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研究生:王嘉琳
研究生(外文):Chia-Lin Wang
論文名稱:誘導生成反義股來抑制斑馬魚心臟肌鈣蛋白的表現
論文名稱(外文):Knock Down the Expression of Zebrafish Cardiac Troponin C in Heart by Conditionally Generating Antisense Strand in vivo
指導教授:蔡懷楨蔡懷楨引用關係
指導教授(外文):Huai-Jen Tsai
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:漁業科學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:53
中文關鍵詞:肌鈣蛋白心臟斑馬魚反義股
外文關鍵詞:Hearttet-on systemcTnCtroponin Czebrafish
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目前斑馬魚中要以弁鈳鄍?lose-of-function)的方式來瞭解基因弁遄A是藉由注射Morpholino (MO)來降低(knock down)蛋白質的轉譯(translation),但MO必須在胚胎剛開始發育的一至四細胞期(1-cell to 4-cell stage)便要立即注射,因此對於要研究基因在胚胎發育晚期甚至成體時的弁鄎K不適用。本實驗則是利用tet-on調控系統配合心臟專一表現基因(cardiac myosin light chain 2)的啟動子(promoter),在斑馬魚胚胎於受精後12小時(12 hours post-fertilization)加入四環黴素衍生物Doxycycline (Dox)誘導產生活化子(transactivator)之後,和Dox結合而驅動下游雙向啟動子(bi-directional promoter)兩側的cTnC-antisense mRNA與綠色螢光報導基因(EGFP)在心臟專一的表現,用以研究心肌肌鈣蛋白(cardiac troponin C, cTnC)的弁遄C結果發現受精後第6天在產生cTnC-antisense mRNA之轉殖魚品系其平均心跳數顯著慢於控制組轉殖魚的心跳數(cTnC-antisense 150.2下/每分鐘;控制組193.7 下/分鐘);受精後第12天亦然(cTnC-antisense 127.8下/分鐘;控制組168.4 跳/分鐘)。並且經由視訊影像(video image)分析轉殖魚之心室型態,得知在受精後第6天含有cTnC-antisense mRNA之轉殖魚其心室舒張末期體積(End-diastolic volume)有15.4%的比例、心室收縮末期體積(End-systolic volume)有38.5%的比例均超出了控制組轉殖魚的正常範圍,而心室射出分量(ventricular ejection fraction)則有46.2%的比例低於控制組轉殖魚的正常範圍;當受精後第12天,則是有更高比例(分別為60%、50%、90%)的cTnC-antisense轉殖魚具有此種異常症狀。cTnC-antisense轉殖魚所產生心臟異常症狀可由注射低劑量抑制心肌肌鈣蛋白生成的cTnC-MO (0.03375ng)得到類似的心臟病變。心跳節律方面,在受精後第6天的cTnC-antisense轉殖魚中有極少數個體(1.3%)會出現心房心室跳動不對稱現象,此種心律不整類似於人類的不完全房室傳導阻斷(incomplete atrio-ventricular block)疾病。利用RT-PCR可以偵測cTnC-antisense轉殖魚個體在受精後第6天確實有cTnC-antisense mRNA生成。藉由以上證據可知我們可以條件式(conditional)的誘導cTnC-antisense轉殖魚品系,使得心肌肌鈣蛋白部分被抑制後產生心臟病變,而獲知心肌肌鈣蛋白的弁遄A今後,或野i以應用這種含有Tet-on調控系統的轉殖模式魚種從事心臟相關疾病的研究。
The tetracycline-controlled myocardium expression system was used to study zebrafish cardiac troponin C (cTnC), a calcium binding protein specifically expressed in heart. GFP reporter and antisense strand of cTnC were constructed on both side of the bi-directional promoter, which was response to the doxycycline (Dox)-induced transactivator driven by a heart –specific promoter of cardiac myosin light chain 2. GFP reporter gene was specifically expressed in the heart of transgenic fish F1 when 10μg/ml of Dox was continuously treated the 12-hpf embryos. We found that the average heart-beating rate of cTnC-antisense transgenic fish was significantly slower than that of the control fish at 6-dpf (150.2 vs. 193.7 beats per min) and 12-dpf (127.8 vs. 168.4 beats per min). The transcription of antisese mRNA of cTnC was detected by RT-PCR at 6-dpf. Furthermore, ventricle morphology was measured from video images of the 6-dpf and 12-dpf embryos in order to compare the difference in term of ventricular function between the control and cTnC-antisense transgenic fish. The end-diastolic and end-systolic volumes of the ventricle of the latter fish were increased, while the ventricular ejection fractions were decreased. Embryos that were injected with low dosage (0.03375ng) of cTnC-morpholino (MO) photocopied the defects of the cTnC-antisense transgenic fish. An asymmetric heart-beat between atrium and ventricle, a syndrome that is similar to the human incomplete atrio- ventricular blocking disease, was observed in few induced hearts (1.3%). These evidences demonstrate that the heart-specific protein is capable of being partially inhibited to translate in the transgenic zebrafish, suggesting this tet-on system can be potentially applied in studying heart disease.
中文摘要 ---------------------------------------------- 2

英文摘要 ---------------------------------------------- 4

前言 -------------------------------------------------- 5

材料與方法 -------------------------------------------- 9

結果 ------------------------------------------------- 17

討論 ------------------------------------------------- 26

參考文獻 --------------------------------------------- 34

圖表 ------------------------------------------------- 37

附錄 ------------------------------------------------- 52
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