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標題: | 利用基因轉殖魚研究cardiac troponin C異常對心臟功能的影響 Effect of the Truncated cTnC Expression on Cardiac Function in Transgenic Fish |
作者: | Chun-An Pai 白峻安 |
指導教授: | 蔡懷楨 |
關鍵字: | 斑馬魚,心肌症,心臟, zebrafish,Tet-on,cardiomyopathy, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | Cardiac troponin C (cTnC)突變可能造成心肌症(cardiomyopathy),其是老年人最嚴重的心臟疾病之一。可是,用來研究在晚期發生心臟失調的模式動物十分缺乏。斑馬魚是個良好的研究材料,因為胚胎透明且利用Tet-on在in vivo下誘導系統已經建立。所以本研究先建立可誘導的基因轉殖斑馬魚 (D12)品系。這個品系是轉殖線性化的pICMLM2-BI-cTnCII-EK經篩選及育養所得到的F2子代。這些子代所衍生的胚胎以doxycycline (Dox,10 μg/ml)處理,會同時表現心臟專一的綠色螢光蛋白(EGFP)和truncated form (去除calcium-binding motif I及II)的cTnC。當我們誘導受精後3天的胚胎,我們觀察到在誘導後第4天,於可誘導產生truncated form cTnC之轉殖魚(D12)的心跳數(188.9± 21.7 下/分鐘)低於只含有pICMLE之轉殖魚控制組(A34) ( 199.8±22.2 下/分鐘)。而且在Dox誘導後第1到6天發現可誘導產生truncated form cTnC轉殖魚(D12)之心室舒張體積和心室收縮體積都大於控制組;更進一步,發現可誘導產生truncated form cTnC轉殖魚(D12)的心室射出分量也都低於控制組,尤其在誘導後第5天(D12為41.9±6.8 % ;control A34為49.3±5.7 %)。另外一方面,大部分誘導產生truncated form cTnC之轉殖魚(D12)個體在誘導後6到9天,心房會逐漸膨大,且有些個體在晚期心房會忽然縮小,之後很快就死亡。同時也發現,在可誘導產生truncated form cTnC之轉殖魚(D12)的心室心肌壁有厚薄不均的現象(5.4∼24.3μm),這個症狀類似人類dilated cardiomyopathy的eccentric hypertrophy現象。總之,誘導產生truncated form cTnC在心臟專一表現之後,會造成和正常cTnC的活性競爭導致轉殖魚心
室舒張體積增加、收縮體積增加、心室射出分量減少和心肌壁厚薄不均,而這些都符合dilated cardiomyopathy的病徵。所以我們認為利用可誘導產生truncated form cTnC之轉殖魚(D12)對於心肌症研究是個非常有潛力的模式動物。 Mutations of cardiac troponin C (cTnC) may cause cardiomyopathy, which is one of the most serious heart diseases of elders, but there is no suitable animal model to study this late-onset disorder. Zebrafish is a good material due to its embryo is transparent and the inducible system in vivo is developed. Zebrafish inducible transgenic line was carried out which expressed cardiac specific EGFP and truncated cTnC at the same time with doxcycline (10μg/ml) induction. After induction at 3 dpf of F2 embryo, we observed that the heart beating rate of zebrafish inducible transgenic line was slower than that of the control fish (188.9± 21.7 Vs. 199.8±22.2 beats/min) after 4 days induction. Both the diastolic volume and the systolic volume of zebrafish inducible transgenic line were larger than control group over the observation time (after 1 to 6 days induction). Furthermore, the ejection fraction of zebrafish inducible transgenic line was also lower than control group during the observation period (after 1 to 6 days induction), especially at 5 days induction (8 dpf) (41.9±6.8 % Vs. 49.3±5.7 %).On the other hand, most of the individuals revealed progressively dilated atrium syndrome till 6-9 days induction, and the individuals with syndrome would die soon. And we found the myocardium wall of ventricle of zebrafish inducible transgenic line (D12) have ventricular muscle wall irregular thickening(5.4∼24.3μm).This phenotype is like the eccentric hypertrophy of human dilated cardiomyopathy. In conclusion, induction of heart-specific truncated form cTnC competing with normal cTnC activity would lead to increased end-diastolic ventricular volume、increased end-systolic ventricular volume、decreased ejection fraction and heart wall thickness disproportionate,these all are the symptom of dilated cardiomyopathy. We think that zebrafish inducible transgenic line is a potential animal model for cardiomyopathy research. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25648 |
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顯示於系所單位: | 分子與細胞生物學研究所 |
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