本篇論文分為兩部分，第一部份是針對突變種品系的斑馬魚，發現在早期發育時期有脊索發育不良並嚴重的扭曲情形，經過銅離子浸泡後，脊索的變異程度有漸緩的趨勢，這樣的結果可能與銅離子代謝有關。實驗中收集了5 天大的斑馬魚胚胎，在冷凍切片後，用蘇木精與伊紅染色 ( Hematoxylin and Eosin stain ) 結果發現，在脊索部位有增生的細胞且細胞核不正常的分布，再利用癌化的標定抗體 PCNA 與 DAPI 細胞核的標定物質的雙抗染下，證實脊索附近細胞增生的情形，而更進ㄧ步的研究發現變異的部位，經過 F59 肌肉的標記抗體與 DAPI 的螢光雙抗染下，並非肌肉組織群細胞，更是符合在原位雜交的實驗上，呈現的證據證明脊索部位隆起不正常的細胞並非肌肉組織，而是脊索內部癌化細胞，而在養育ㄧ個月時期的斑馬魚發現硬骨染色下，骨骼不正常排列情形。基於上述實驗可發現本篇論文與ㄧ種罕見的遺傳性疾病－ Menkes Disease 非常類似，此疾病是因為銅離子代謝失常造成疾病。在實驗中，培育了脊索突變種品系斑馬魚在發育初期脊索部位、肌肉組織甚至皮膚組織，均有嚴重的變異與細胞核增多癌化的情形，在此品系中我們可以作為人類 Menkes 症候群的模式動物研究。 第二部份則是針對於建立 CKII-E7-RFP 紅螢光品系的斑馬魚，在表皮的表現增生、癌化的情形。根據以上的實驗，我們更是可充分發現以斑馬魚當作脊索骨骼與皮膚的模式物種，應用於基因與藥物的研究上為相當有利實驗方式。 These are two subjects in this thesis. In the first subject, we identified a novel notochord mutant zebrafish strain that has malformed and seriously twisted notochords, but these morphological changes can be attenuated by exposure with copper metabolism. Histologically, there are many cells with high N/C ratio and pleomorphic nuclears around the malformed notochord region. In order to further investigate the molecular mechanisms underlying the malformed and seriously twisted notochord, we stained the 5-dpf embryos with antibodies PCNA, F59,and follows by cryosectioning and counter stained with DAPI. Results showed that the malformed and seriously twisted notochord regions are due to notochord but not muscle cells growth dysplasia. In addition, the bones of juvenile (~one month old) are disorganized growth. These phenotypic changes and molecular evidences are highly similar to a human hereditary disease, Menkes disease. In the second subject, we established a CHII-E7-RFP transgenic zebrafish line in order to study the molecular mechanism of E7 oncoprotein and skin cancer, In conclusion, this notochord mutant zebrafish strain and CKII-E7-RFP transgenic zerbrafish line could be used as an animal model to study Menkes disease and skin cancer,respectively.
