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系統識別號 U0002-0408200811343400
中文論文名稱 利用斑馬魚來研究皮膚及脊索病變
英文論文名稱 Using zebrafish as a model to study notochord and epidermis growth disorganization
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 96
學期 2
出版年 97
研究生中文姓名 林己勝
研究生英文姓名 Ji-Sheng Lin
學號 695180132
學位類別 碩士
語文別 中文
第二語文別 英文
口試日期 2008-07-18
論文頁數 61頁
口試委員 指導教授-陳曜鴻
委員-林明德
委員-鄭邑荃
中文關鍵字 緬克斯症候群  斑馬魚 
英文關鍵字 Menkes disease  zebrafish 
學科別分類 學科別醫學與生命科學生物學
中文摘要 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。
英文摘要 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.
論文目次 中文摘要 ………………………………………………………………………I
英文摘要 …………………………………………………………………… III
目 錄 ……………………………………………………………………IV
圖、表目錄 …………………………………………………………………… V
第一部份:利用斑馬魚來研究脊索病變
一、前言………………………………………………………………………1
二、材料與方法……………………………………………………………… 7
三、結果 …………………………………………………………………… 17
四、討論 ……………………………………………………………………24
五、圖、表…………………………………………………………………… 41
第二部份:利用斑馬魚來研究皮膚病變
一、前言 …………………………………………………………………… 29
二、材料與方法 …………………………………………………………… 33
三、結果與討論 ……………………………………………………………39
四、圖、表 …………………………………………………………………53
參考文獻 ………………………………………………………………………55


圖、表 目錄
第一部份圖、表
圖 1:在斑馬魚胚胎中脊索病變使其脊索組織部位呈現波浪狀的顯徵變異情
形 ………………………………………………………………………………41
圖 2:Type I 各個不同時期斑馬魚所呈現脊索絮亂情形 …………………42
圖 3:Type II 斑馬魚時期在脊索的尾端有腫瘤的產生與野生種的斑馬魚相
互比較下的情形 ………………………………………………………………43
圖 4:Type I 與 Type II 突變種斑馬魚的觀察病理切片 ………………… 44
圖 5:突變種斑馬魚病理切片 PCNA 與 DAPI 雙抗染的情形 ………… 45
圖 6:突變種斑馬魚病理切片 PCNA 與 Hematoxylin 雙抗染的情形 … 46
圖 7:突變種斑馬魚病理切片 F59 與 DAPI 雙抗染的情形 …………… 47
圖 8:Co2al 與 MyoD in situ hybrdiziation 在脊索的表現的訊號圖……… 48
圖 9:Co2al 在脊索部位表現的切片情形 ………………………………… 49
圖 10:突變種品系斑馬魚浸泡銅離子變異折線圖…………………………50
圖 11:突變種斑馬魚的硬骨染色情形……………………………………… 51
圖 12:脊索病變的斑馬魚品系致病的可能路徑……………………………52
表 1:突變種斑馬魚品系的鑑定與統計………………………………………54





第二部份圖、表
圖 13:CKII-E7-RFP 紅螢光品系的斑馬魚 (F0) 與 PCNA 的抗染結
果……………………………………………………………………………… 53
圖 14:CKII-E7-RFP 紅螢光品系的斑馬魚 (F1) 紅螢光表現情形……… 53
表 2:CII-E7-RFP 轉殖魚品系的鑑定與統計……………………………… 54

參考文獻 中華民國人類遺傳學會,疾病資料庫,緬克斯症候群
行政院衛生署國民健康局遺傳疾病諮詢服務
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