建立用於探討心肌梗塞與其治療發展的整合性斑馬魚研究平台

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研究生：	許家豪
論文名稱：	建立用於探討心肌梗塞與其治療發展的整合性斑馬魚研究平台 An integrated zebrafish-based assay platform for the study of myocardial infarction and therapeutics development
指導教授：	莊永仁
口試委員:	吳長益蘇士哲莊永仁
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	42
中文關鍵詞：	斑馬魚、心肌梗塞、藥物發展平台
相關次數：	點閱：2 下載：0
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隨著人口結構的老化以及肥胖人口的增加,心血管疾病對人類的健康傷害極大其致死率甚至超過癌症.其中,又以心肌梗塞占大多數,病患因冠狀動脈阻塞造成其下游的心肌細胞壞死,最終因心律不整而死亡.至目前為止,臨床上仍未能找出有效的方法來促進受損的心臟恢復.斑馬魚是近年來非常受歡迎的疾病模式生物,其優點包含胚胎上的透明性,能產生大的子代,已被解碼的基因遺傳訊息與人類有高度的相似性,並且有大量文獻使用斑馬魚來探討心血管相關疾病.在近期心臟再生的研究上,斑馬魚展現其獨特的修復能力,正因此項特性,我們希望利用斑馬魚來建立用於探討心肌梗塞與其治療發展的整合性平台.
在本篇研究,可分為四個部分.第一部份,我們建立一個心肌梗塞的斑馬魚模式用於探討心臟受損及修復的過程,並從細胞的層次進行觀測.第二部分,我們測試一套新穎的斑馬魚心電圖系統,用於量測斑馬魚受損之心臟電生理狀況.第三部分,我們結合血管造影技術及螢光轉殖基因魚,來探討斑馬魚受損之心血管循環恢復情形.第四部份,我們製備新的螢光轉殖基因魚,期望應用於藥物檢測.最終,我們希望整合這些平台並提供心肌梗塞上的研究與其治療方向.

Table of contents
中文摘要    I
Abstract    II
致謝    IV
Abbreviations    VII
1.    Introduction    1
  1.1    The cardiovascular system and its diseases    1
  1.2    Zebrafish model for myocardial infarction    2
  1.3    Cardiac regenerative capacity in zebrafish    3
  1.4    Important events during cardiac repair in zebrafish    4
  1.5    Advantages of zebrafish model for myocardial infarction research and therapeutics development    5
  1.6    The objective of this study    6
2.    Material and Methods    8
  2.1    Zebrafish strains and transgenic fish lines stocks    8
  2.2    Generation of new transgenic fish line    8
  2.3    Cryoinjury    8
  2.4    Histology and immunofluorescence staining    9
  2.5    Angiography in adult zebrafish- retro orbital injection    9
  2.6    Image capture and the use of microscope    10
3.    Results    11
  3.1    The adult zebrafish model of cryoinjury-induced myocardial infarction    11
3.1.1    Cryoinjury-induced myocardial infarction in zebrafish    11
3.1.2    Morphological change in the ventricle apex in response to cryoinjury    12
3.1.3    Extent of tissue damage following cryoinjury    12
3.1.4    Cryoinjury induces phagocytes recruitment in the lesion area    14
3.2    Assessing the extent of cardiac injury by electrocardiography    15
3.2.1    Functional analysis by a custom-made ECG kit and a digital camera    15
3.2.2    Prolonged ventricular re-polarization caused by infarction in zebrafish heart    16
3.3    Endothelial dynamics in response to myocardial infarction    17
3.3.1    Rapid recovery of the blood vessel endothelium in the infarcted ventricle    18
3.3.2    Blood reperfusion within the infarction site    19
3.4    Generation of transgenic zebrafish to report heart regeneration    20
3.4.1    Generation of Tg(gata4:nls-mCherry/cmlc2:nls-EGFP) to report cardiomyocytes dedifferentiation in real time    21
3.4.2    F1 generation of Tg(gata4:nls-mCherry/cmlc2:nls-EGFP) were identified by two-color fluorescent signals in the heart.    21
4.    Discussion    23
  4.1    Requirement of assay platform for the study of MI-like injury in zebrafish model    24
  4.2    Endothelial dynamics – vessel re-growth and blood reperfusion    25
  4.3    Application for drug screen    26
  4.4    Future perspective    27
5.    Reference    28

List of Figures    31
Fig1. Morphological change in the ventricle apex in response to cryoinjury    31
Fig2. Necrotic or apoptotic cells in the ventricle apex of cryoinjured heart    32
Fig3. Cryoinjury leads to loss of endothelial cells and cardiomyocytes.    33
Fig4. Phagocytes were recruited to the lesion site following cryoinjury    34
Fig5. Prolonged ventricular re-polarization caused by cryoinjury-induced infarction in zebrafish isolated heart.    35
Fig6. Rapid recovery of the cardiac endothelium in the infarction site upon cryoinjury    36
Fig7. Infarction region rapidly vascularized and re-perfused after cryoinjury    37
Fig8.  Stable transgenic zebrafish line Tg(gata4:nls-mCherry/cmlc2:nls-EGFP) were identified by two-color fluorescent signals in the heart    38

List of supporting information    39
FigS1. Preparation of cryoprobe    39
  FigS2. Phagocytes retained in the apex of ventricle at 7dpc following cryoinjury………..40
FigS3. Combination of a custom-made ECG kit and a digital camera for the assessment of heart function    41
FigS4. Tail vein vasculature and blood flow could be detected by angiography    42



                                

1. Bui AL, Horwich TB, Fonarow GC (2011) Epidemiology and risk profile of heart failure. Nat Rev Cardiol 8: 30-41.
2. van den Borne SW, Diez J, Blankesteijn WM, Verjans J, Hofstra L, et al. (2010) Myocardial remodeling after infarction: the role of myofibroblasts. Nat Rev Cardiol 7: 30-37.
3. Ptaszek LM, Mansour M, Ruskin JN, Chien KR (2012) Towards regenerative therapy for cardiac disease. Lancet 379: 933-942.
4. Garbern JC, Mummery CL, Lee RT (2013) Model systems for cardiovascular regenerative biology. Cold Spring Harb Perspect Med 3: a014019.
5. Ou L, Li W, Liu Y, Zhang Y, Jie S, et al. (2010) Animal models of cardiac disease and stem cell therapy. Open Cardiovasc Med J 4: 231-239.
6. Hasenfuss G (1998) Animal models of human cardiovascular disease, heart failure and hypertrophy. Cardiovasc Res 39: 60-76.
7. Gonzalez-Rosa JM, Martin V, Peralta M, Torres M, Mercader N (2011) Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development 138: 1663-1674.
8. Chablais F, Veit J, Rainer G, Jazwinska A (2011) The zebrafish heart regenerates after cryoinjury-induced myocardial infarction. BMC Dev Biol 11: 21.
9. Schnabel K, Wu CC, Kurth T, Weidinger G (2011) Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS One 6: e18503.
10. Lien CL, Harrison MR, Tuan TL, Starnes VA (2012) Heart repair and regeneration: recent insights from zebrafish studies. Wound Repair Regen 20: 638-646.
11. Frangogiannis NG (2006) The mechanistic basis of infarct healing. Antioxid Redox Signal 8: 1907-1939.
12. Poss KD, Wilson LG, Keating MT (2002) Heart regeneration in zebrafish. Science 298: 2188-2190.
13. Laflamme MA, Murry CE (2011) Heart regeneration. Nature 473: 326-335.
14. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe-Heider F, et al. (2009) Evidence for cardiomyocyte renewal in humans. Science 324: 98-102.
15. Hsieh PC, Segers VF, Davis ME, MacGillivray C, Gannon J, et al. (2007) Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 13: 970-974.
16. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, et al. (2011) Transient regenerative potential of the neonatal mouse heart. Science 331: 1078-1080.
17. Mahmoud AI, Porrello ER (2012) Turning back the cardiac regenerative clock: lessons from the neonate. Trends Cardiovasc Med 22: 128-133.
18. Oberpriller JO, Oberpriller JC, Matz DG, Soonpaa MH (1995) Stimulation of proliferative events in the adult amphibian cardiac myocyte. Ann N Y Acad Sci 752: 30-46.
19. Raya A, Koth CM, Buscher D, Kawakami Y, Itoh T, et al. (2003) Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proc Natl Acad Sci U S A 100 Suppl 1: 11889-11895.
20. Poss KD (2007) Getting to the heart of regeneration in zebrafish. Semin Cell Dev Biol 18: 36-45.
21. Kikuchi K, Holdway JE, Werdich AA, Anderson RM, Fang Y, et al. (2010) Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes. Nature 464: 601-605.
22. Kikuchi K, Holdway JE, Major RJ, Blum N, Dahn RD, et al. (2011) Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration. Dev Cell 20: 397-404.
23. Lafontant PJ, Burns AR, Grivas JA, Lesch MA, Lala TD, et al. (2012) The giant danio (D. aequipinnatus) as a model of cardiac remodeling and regeneration. Anat Rec (Hoboken) 295: 234-248.
24. Nagaya N, Fujii T, Iwase T, Ohgushi H, Itoh T, et al. (2004) Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis. Am J Physiol Heart Circ Physiol 287: H2670-2676.
25. Kuraitis D, Ruel M, Suuronen EJ (2011) Mesenchymal stem cells for cardiovascular regeneration. Cardiovasc Drugs Ther 25: 349-362.
26. Steinhauser ML, Lee RT (2011) Regeneration of the heart. EMBO Mol Med 3: 701-712.
27. Martin-Puig S, Fuster V, Torres M (2012) Heart repair: from natural mechanisms of cardiomyocyte production to the design of new cardiac therapies. Ann N Y Acad Sci 1254: 71-81.
28. Tan JL, Zon LI (2011) Chemical screening in zebrafish for novel biological and therapeutic discovery. Methods Cell Biol 105: 493-516.
29. Ali S, Champagne DL, Spaink HP, Richardson MK (2011) Zebrafish embryos and larvae: a new generation of disease models and drug screens. Birth Defects Res C Embryo Today 93: 115-133.
30. Shafizadeh E, Huang H, Lin S (2002) Transgenic zebrafish expressing green fluorescent protein. Methods Mol Biol 183: 225-233.
31. Chablais F, Jazwinska A (2012) Induction of myocardial infarction in adult zebrafish using cryoinjury. J Vis Exp.
32. Gonzalez-Rosa JM, Mercader N (2012) Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish. Nat Protoc 7: 782-788.
33. Huang WC, Hsieh YS, Chen IH, Wang CH, Chang HW, et al. (2010) Combined use of MS-222 (tricaine) and isoflurane extends anesthesia time and minimizes cardiac rhythm side effects in adult zebrafish. Zebrafish 7: 297-304.
34. Pugach EK, Li P, White R, Zon L (2009) Retro-orbital injection in adult zebrafish. J Vis Exp.
35. Ecc Committee S, Task Forces of the American Heart A (2005) 2005 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation 112: IV1-203.
36. Blankesteijn WM, Creemers E, Lutgens E, Cleutjens JP, Daemen MJ, et al. (2001) Dynamics of cardiac wound healing following myocardial infarction: observations in genetically altered mice. Acta Physiol Scand 173: 75-82.
37. Couffinhal T, Dufourcq P, Barandon L, Leroux L, Duplaa C (2009) Mouse models to study angiogenesis in the context of cardiovascular diseases. Front Biosci 14: 3310-3325.
38. Itou J, Oishi I, Kawakami H, Glass TJ, Richter J, et al. (2012) Migration of cardiomyocytes is essential for heart regeneration in zebrafish. Development 139: 4133-4142.
39. Suster ML, Kikuta H, Urasaki A, Asakawa K, Kawakami K (2009) Transgenesis in zebrafish with the tol2 transposon system. Methods Mol Biol 561: 41-63.
40. Chablais F, Jazwinska A (2012) The regenerative capacity of the zebrafish heart is dependent on TGFbeta signaling. Development 139: 1921-1930.
41. Kikuchi K, Gupta V, Wang J, Holdway JE, Wills AA, et al. (2011) tcf21+ epicardial cells adopt non-myocardial fates during zebrafish heart development and regeneration. Development 138: 2895-2902.
42. Stroupe KT, Morrison DA, Hlatky MA, Barnett PG, Cao L, et al. (2006) Cost-effectiveness of coronary artery bypass grafts versus percutaneous coronary intervention for revascularization of high-risk patients. Circulation 114: 1251-1257.
43. Cao Y, Semanchik N, Lee SH, Somlo S, Barbano PE, et al. (2009) Chemical modifier screen identifies HDAC inhibitors as suppressors of PKD models. Proc Natl Acad Sci U S A 106: 21819-21824.
44. Blackburn JS, Liu S, Raimondi AR, Ignatius MS, Salthouse CD, et al. (2011) High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope. Nat Protoc 6: 229-241.

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