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.

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

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 
C Nüsslein-Volhard, R Dahm, Zebrafish, A Practical Approach, 2002 Lewis J . Kleinsmith. Principle of cancer biology Abdelilah, Zehava Rangini4, Elizabeth Mountcastle-Shah and Wolfgang Driever.  Mutations  affecting  development  of  the  notochord  in  zebrafish. 
Development 123, 117-128   
Derek L. Stemple, Lilianna Solnica-Krezel, Fried Zwartkruis1, Stephan  C.  F.  Neuhauss,  Alexander  F.  Schier2,  Jarema  Malicki,  Didier  Y.  R. 
Stainier3, Salim Abdelilah, Zehava Rangini4,Elizabeth Mountcastle- Shah  and  Wolfgang  Driever.  Mutations  affecting  development  of  the notochord in zebrafish    Development 123, 117-128. 
John Doorbar, The papillomavirus life cycle. Journal of Clinical Virology 32S (2005) S7–S15. 
Jörg Odenthal, Pascal Haffter, Elisabeth Vogelsang, Michael Brand, Fredericus  J.  M.  van  Eeden,  Makoto  Furutani-Seiki,  Michael  Granato, Matthias Hammerschmidt, Carl-Philipp Heisenberg, Yun-Jin Jiang, Donald 
A. Kane, Robert N. Kelsh, Mary C. Mullins, Rachel M. Warga, Miguel L. Allende,  Eric  S.  Weinberg  and  Christiane  Nüsslein-Volhard    Mutations affecting  the  formation  of  the  notochord  in  the  zebrafish,  Danio  rerio  
Akgül B, Cooke JC, Storey AHPV-associated skin disease. J Pathol. 2006 Jan;208(2):165-75. 
Anderson C, Bartlett SJ, Gansner JM, Wilson D, He L, Gitlin JD, Kelsh RN, Dowden J. Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis. Mol Biosyst. 2007 Jan;3(1):51-9   
Bernard HU. The clinical importance of the nomenclature, evolution and taxonomy of human papillomaviruses. J Clin Virol 2005;32S:S1–S6 
Bowden GT. Prevention of non-melanoma skin cancer by targeting ultraviolet-B-light signalling. Nat. Rev. Cancer 2004; 4:23–35. 
Butler E, Hardin J, Benson S. The role of lysyl oxidase and collagen crosslinking   during   sea   urchin   development.   Exp   Cell   Res.   1987 Nov;173(1):174-82. 
Chelly J, Tümer Z, Tønnesen T, Petterson A, Ishikawa-Brush Y, Tommerup N, Horn N, Monaco AP. Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat Genet. 1993 Jan;3(1):14-9.   
Dahle J, Kvam E, Stokke T. Bystander effects in UV-induced genomic   instability: antioxidants inhibit delayed mutagenesis induced by ultraviolet A and B radiation. J Carcinog. 2005 Aug 9;4:11.   
Danks DM, Campbell PE, Stevens BJ, Mayne V, Cartwright E.Menkes's kinky hair syndrome. An inherited defect in copper absorption with widespread effects. Pediatrics. 1972 Aug;50(2):188-201. 
DePinho RA.The age of cancer. Nature. 2000 Nov 9;408(6809):248-54. Review. 
Di Donato A, Lacal JC, Di Duca M, Giampuzzi M, Ghiggeri G, Gusmano  R.Micro-injection of recombinant lysyl oxidase blocks oncogenic p21-Ha-Ras and progesterone effects on Xenopus laevis oocyte maturation. FEBS Lett. 1997 Dec 8;419(1):63-8. 
Dimaculangan DD, Chawla A, Boak A, Kagan HM, Lazar MA. 
Dong W, Kloz U, Accardi R, Caldeira S, Tong WM, Wang ZQ, Jansen L, Dürst M, Sylla BS, Gissmann L, Tommasino M. Skin hyperproliferation 
and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38. J Virol. 2005 Dec;79(23):14899-908. 
Durham SE, Krishnan KJ, Betts J, Birch-Machin MA. Mitochon- drial DNA damage in non-melanoma skin cancer. Br J Cancer 2003; 88:90–95. 
Essential role of lysyl oxidases in notochord development. Dev Biol. 2007 Jul 15;307(2):202-13. 
Friedman RM, Yeh A, Gutman P, Contente S, Kenyon K. Reversion by deletion of transforming oncogene following interferon-beta and retinoic 
acid treatment. J Interferon Cytokine Res. 1997 Oct; 
17(10):647-51. 
Gansner JM, Mendelsohn BA, Hultman KA, Johnson SL, Gitlin JD.Goka TJ, Stevenson RE, Hefferan PM, Howell RR.Menkes disease: a biochemical abnormality in cultured human fibroblasts. Proc Natl Acad Sci U S A. 1976 Feb;73(2):604-6. 
Hawkins RL, Fan J, Hille MB. Gastrulation in the sea urchin, Strongylocentrotus purpuratus, is disrupted by the small laminin peptides YIGSR and IKVAV. Cell Adhes Commun. 1995 May;3(2):163-77. 
Haendel MA, Tilton F, Bailey GS, Tanguay RL. Developmental toxicity of the dithiocarbamate pesticide sodium metam in zebrafish. Toxicol Sci. 2004 Oct;81(2):390-400. 
Hawk SN, Uriu-Hare JY, Daston GP, Jankowski MA, Kwik-Uribe C, Rucker RB, Keen CL. Rat embryos cultured under copper-deficient conditions develop abnormally and are characterized by an impaired oxidant defense system. Teratology. 1998 Jun;57(6):310-20. 
Hay ED. Extracellular matrix, cell skeletons, and embryonic development. Am J Med Genet. 1989 Sep;34(1):14-29. 
Jablonska S, Dabrowski J, Jakubowicz K. Epidermodysplasia   
verruciformis as a model in studies on the role of papovaviruses in oncogenesis. Cancer Res. 1972 Mar;32(3):583-9. 
Jankowski MA, Uriu-Hare JY, Rucker RB, Rogers JM, Keen CL. Maternal zinc deficiency, but not copper deficiency or diabetes, results in increased embryonic cell death in the rat: implications for mechanisms 
underlying abnormal development. Teratology. 1995 Feb;51(2):85-93. 
Jankowski MA, Uriu-Hare JY, Rucker RB, Keen CL. Effect of maternal diabetes and dietary copper on fetal development in rats. Reprod Toxicol. 1993 Nov-Dec;7(6):589-98. 
Kenyon K, Modi WS, Contente S, Friedman RM. A novel human cDNA with a predicted protein similar to lysyl oxidase maps to chromosome 15q24-q25. J Biol Chem. 1993 Sep 5;268(25):18435-7. 
Kuo YM, Gitschier J, Packman S. Developmental expression of the mouse mottled and toxic milk genes suggests distinct functions for the Menkes and Wilson disease copper transporters. Hum Mol Genet. 1997 Jul;6(7):1043-9.   
Leonardi E, Girlando S, Serio G, Mauri FA, Perrone G, Scampini S,   Dalla Palma P, Barbareschi M. PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables. J Clin Pathol. 1992 May;45(5):416-9. 
Levinson B, Vulpe C, Elder B, Martin C, Verley F, Packman S, Gitschier J. The mottled gene is the mouse homologue of the Menkes disease gene. Nat Genet. 1994 Apr;6(4):369-73.   
Madsen EC, Morcos PA, Mendelsohn BA, Gitlin JD.In vivo correction of a Menkes disease model using antisense oligonucleotides. Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3909-14. 
Mendelsohn BA, Yin C, Johnson SL, Wilm TP, Solnica-Krezel L, Gitlin JD. Atp7a determines a hierarchy of copper metabolism essential for notochord development. Cell Metab. 2006 Aug; 4(2):155-62. 
Mantovani F, Banks L. The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene. 2001 Nov 26;20(54):7874-87.   
Menkes JH. Kinky hair disease: twenty five years later. Brain Dev. 1988;10(2):77-9.   
Mercer JF, Grimes A, Ambrosini L, Lockhart P, Paynter JA, Dierick H, Glover TW. Mutations in the murine homologue of the Menkes gene in dappled and blotchy mice. Nat Genet. 1994 Apr;6(4):374-8.   
Mercer JF, Livingston J, Hall B, Paynter JA, Begy C, Chandrasekharappa S, Lockhart P, Grimes A, Bhave M, Siemieniak D, et al. Isolation of a partial candidate gene for Menkes disease by positional cloning. Nat Genet. 1993 Jan;3(1):20-5.   
Miller DL, Weinstock MA.Nonmelanoma skin cancer in the United States: incidence. J Am Acad Dermatol. 1994 May;30(5 Pt 1):774-8. 
Münger K, Basile JR, Duensing S, Eichten A, Gonzalez SL, Grace M, Zacny VL. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene. 2001 Nov 26;20(54):7888-98.   
Münger K, Baldwin A, Edwards KM, Hayakawa H, Nguyen CL, Owens M, Grace M, Huh K.Mechanisms of human papillomavirus-induced oncogenesis. J Virol. 2004 Nov; 78(21):11451-60.   
Parker SL, Tong T, Bolden S, Wingo PA.Cancer statistics, 1996.CA   
Cancer J Clin. 1996 Jan-Feb;46(1):5-27.   
Pfister H. Chapter 8: Human papillomavirus and skin cancer. 
J Natl Cancer Inst Monogr. 2003;(31):52-6. Review. 
Proud VK, Mussell HG, Kaler SG, Young DW, Percy AK. Distinctive Menkes disease variant with occipital horns: delineation of natural history and clinical phenotype. Am J Med Genet. 1996 Oct 2;65(1):44-51. 
Retinoic acid prevents downregulation of ras recision gene/lysyl oxidase early in adipocyte differentiation. Differentiation. 1994 Nov;58(1):47-52. 
Reiser K, McCormick RJ, Rucker RB. Enzymatic and nonenzymatic cross-linking of collagen and elastin. FASEB J. 1992 Apr;6(7):2439-49.   
Sander CS, Hamm F, Elsner P, Thiele JJ. Oxidative stress in malignant melanoma and non-melanoma skin cancer. Br J Dermatol. 2003 May;148(5):913-22. 
Schaper ID, Marcuzzi GP, Weissenborn SJ, Kasper HU, Dries V, Smyth N, Fuchs P, Pfister H. Development of skin tumors in mice transgenic for early genes of human papillomavirus type 8. Cancer Res. 2005 Feb 15;65(4):1394-400.   
Snijders PJ, Steenbergen RD, Heideman DA, Meijer CJ. HPV-mediated cervical carcinogenesis: concepts and clinical implications. J Pathol. 2006 Jan;208(2):152-64.   
Stern RS. The mysteries of geographic variability in nonmelanoma skin cancer incidence. Arch Dermatol. 1999 Jul;135(7):843-4. 
Tan RS, Taniguchi T, Harada H. Identification of the lysyl oxidase gene as target of the antioncogenic transcription factor, IRF-1, and its possible role in tumor suppression. Cancer Res. 1996 May 15;56(10):2417-21. 
Tinsley JM, Fisher C, Searle PF. Abnormalities of epidermal differentiation associated with expression of the human papillomavirus type 1 early region in transgenic mice. J Gen Virol. 1992 May;73 ( Pt 5):1251-60. 
Tümer Z, Vural B, Tønnesen T, Chelly J, Monaco AP, Horn N. Characterization of the exon structure of the Menkes disease gene using vectorette PCR. Genomics. 1995 Apr 10;26(3):437-42. 
Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J. Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat Genet. 1993 Jan;3(1):7-13.   
Wakai S, Ishikawa Y, Nagaoka M, Okabe M, Minami R, Hayakawa T. Central nervous system involvement and generalized muscular atrophy in occipital horn syndrome: Ehlers-Danlos type IX. A first Japanese case. J 
Neurol Sci. 1993 May;116(1):1-5. 
Wessel GM, McClay DR. Gastrulation in the sea urchin embryo requires the deposition of crosslinked collagen within the extracellular matrix. Dev Biol. 1987 May;121(1):149-65. 
zur Hausen H. Papillomavirus infections--a major cause of human cancers. Biochim Biophys Acta. 1996 Oct 9;1288(2):F55-78. Review. 
zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer. 2002 May;2(5):342-50.