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系統識別號 U0026-0201201219060000
論文名稱(中文) 纖維母細胞生長因子-10在胚胎幹細胞分化成心肌細胞中所扮演的角色
論文名稱(英文) The role of fibroblast growth factor-10 in embryonic stem cell derived cardiomyocyte differentiation
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 100
學期 1
出版年 100
研究生(中文) 李惠菁
研究生(英文) Hui-Jing Li
學號 S96994031
學位類別 碩士
語文別 英文
論文頁數 47頁
口試委員 指導教授-謝清河
口試委員-沈家寧
口試委員-黃效民
口試委員-江伯敏
中文關鍵字 纖維母細胞生長因子  胚胎幹細胞  心肌細胞分化 
英文關鍵字 fibroblast growth factors  embryonic stem cells  cardiomyocyte differentiation 
學科別分類
中文摘要 纖維母細胞生長因子(fibroblast growth factors, FGFs)於正常心臟發育扮演著重要的角色。然而尚未有系統性的研究去探討纖維母細胞生長因子是否能增加幹細胞分化成心肌細胞的比例。在本篇研究中,我們專注於探討纖維母細胞生長因子對胚胎幹細胞(ES cells)與誘導性多能幹細胞(iPS cells)進一步增加心肌細胞分化所扮演的角色,並透過實驗來證明纖維母細胞生長因子參與在什麼路徑來調控心肌細胞的分化。
本篇使用的細胞模型為小鼠胚胎幹細胞,此細胞株經轉殖入一段心肌細胞特有的啟動子-粗肌鏈 (αMHC) 連結綠色螢光蛋白的序列,可以透過穩定的表達綠色螢光蛋白來評估心肌細胞分化的比率。首先我們從目前已知的纖維母細胞生長因子重組蛋白中,個別篩選出可能對增加心肌細胞分化比率的成員。在誘導胚胎幹細胞-心肌細胞分化的過程中,只有纖維母細胞生長因子-3, 10, 11, 13和15在brachyury (中胚層標記)和Flk-1(心血管前驅細胞標記)的表現上有較類似的模式。而進一步發現的結果顯示,其中只有纖維母細胞生長因子-10 能有效增加心肌細胞分化。利用纖維母細胞生長因子-10結合抗體中和其作用後發現,心肌細胞分化的比率明顯的被抑制。此抑制效果也可透過加入纖維母細胞生長因子受器的拮抗物PD173074以及纖維母細胞生長因子受器-2 的shRNA而達成。而纖維母細胞生長因子-10除了在胚胎幹細胞中可促進心肌細胞的分化,在iPS 中也能看到相同的效果。在動物實驗中,纖維母細胞生長因子-10結合胚胎幹細胞在打入C57B/L6小鼠心臟時,也可以看到增加胚胎幹細胞分化心肌細胞的效果。
本研究的結果顯示纖維母細胞生長因子-10可以透過結合到胚胎幹細胞和誘導性多能細胞的纖維母細胞生長因子接受器這條路徑,來增加心肌細胞分化比例。希冀此研究在將來可以藉由增加心肌細胞的數目,應用在臨床疾病的治療上。
英文摘要 The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells.
We used mouse ES cells stably transfected with a cardiac-specific a-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time and concentration dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial coadministration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation.
FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications.
論文目次 Abstract I
摘要 II
誌謝 III
TABLE OF CONTENTS IV
TABLE OF FIGURES VI
Chapter 1 Introduction 1
1.1 Heart failure 1
1.2 Urgent need for the new therapy to replace the injured cardiomyocytes 1
1.3 Pluripotency of embryonic stem cells and induced pluripotent stem cells 2
1.4 Obstacles that hinder the development of embryonic stem cell therapy 2
1.5 FGF family is essential for heart development 3
Chapter 2 Materials and methods 5
2.1 Culture of mouse ES-αMHC-EGFP cells 5
2.2 cardiomyocyte differentiation induction 5
2.3 Cell culture of mouse iPS cells 6
2.4 Temporal gene expression profiling 6
2.5 Cardiomyocyte differentiation quantification by flow cytometry analysis 11
2.6 FGF-10 neutralization and FGFR inhibition 12
2.7 Gene knockdown 12
2.8 Immunohistochemical staining 12
2.9 Cell cycle analysis 13
2.10 Preparation of a EBs-peptide nanofiber mixture for transplantation 13
2.11 In vivo evaluation of FGF-10 on cardiomyocyte differentiation. 13
2.12 Microarray analysis 14
2.13 Statistical analysis 14
Chapter 3 Results 15
3.1 Temporal expression profile of FGF members during ES cell cardiomyocyte differentiation 15
3.2 FGF-10 is crucial to cardiomyocyte differentiation from ES cells 16
3.3 Response to FGF-10 is mediated via FGF receptors 17
3.4 The enhancement of cardiomyocyte differentiation by FGF-10 is also apparent in iPS cells 18
3.5 FGF-10 modulates the expression of cardiac related genes and pathways. 19
3.6 FGF-10 enhances cardiomyocyte differentiation from ES cells in vivo 19
Chapter 4 Discussion 40
References 43
參考文獻 1. Jessup M, Brozena S (2003) Heart failure. N Engl J Med 348: 2007-2018.
2. Writing Group Members, Rosamond W, Flegal K, Furie K, Go A, Greenlund K, et al. (2008) Heart disease and stroke statistics--2008 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 117: e 25-146.
3. Jernberg T, Johanson P, Held C, Svennblad B, Lindbäck J, et al. (2011) Association between adoption of Evidence-Based Treatment and Survival for Patients With ST-Elevation Myocardial Infarction. JAMA: The Journal of the American Medical Association 305: 1677-1684.
4. Lichtman JH, Jones SB, Leifheit-Limson EC, Wang Y, Goldstein LB (2011) 30-Day Mortality and Readmission After Hemorrhagic Stroke Among Medicare Beneficiaries in Joint Commission Primary Stroke Center-Certified and Noncertified Hospitals. Stroke 42: 3387-3391.
5. Curry LA, Spatz E, Cherlin E, Thompson JW, Berg D, et al. (2011) What Distinguishes Top-Performing Hospitals in Acute Myocardial Infarction Mortality Rates. Annals of Internal Medicine 154: 384-390.
6. Zuba-Surma EK, Guo Y, Taher H, Sanganalmath SK, Hunt G, et al. (2011) Transplantation of expanded bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) improves left ventricular function and remodelling after myocardial infarction. Journal of Cellular and Molecular Medicine 15: 1319-1328.
7. Caspi O, Huber I, Kehat I, Habib M, Arbel G, et al. (2007) Transplantation of Human Embryonic Stem Cell-Derived Cardiomyocytes Improves Myocardial Performance in Infarcted Rat Hearts. J Am Coll Cardiol 50: 1884-1893.
8. Quevedo HC, Hatzistergos KE, Oskouei BN, Feigenbaum GS, Rodriguez JE, et al. (2009) Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity. Proc Natl Acad Sci U S A 106: 14022-14027.
9. Matsuura K, Honda A, Nagai T, Fukushima N, Iwanaga K, et al. (2009) Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice. J Clin Invest 119: 2204-2217.
10. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, et al. (2007) Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotech 25: 1015-1024.
11. Nelson TJ, Martinez-Fernandez A, Yamada S, Perez-Terzic C, Ikeda Y, et al. (2009) Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells. Circulation 120: 408-416.
12. Takahashi K, Yamanaka S (2006) Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126: 663-676.
13. Segers VF, Lee RT (2008) Stem-cell therapy for cardiac disease. Nature 451:937–942.
14. Takahashi T, Lord B, Schulze PC, Fryer RM, Sarang SS, et al. (2003) Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 107: 1912-1916.
15. Chan SSK, Chen JH, Hwang SM, Wang IR, Li HJ, et al. (2009) Salvianolic acid B-vitamin C synergy in cardiac differentiation from embryonic stem cells. Biochem Biophys Res Commun 387: 723-728.
16. Sadek H, Hannack B, Choe E, Wang J, Latif S, Garry MG, Garry DJ, Longgood J, Frantz DE, Olson EN, Hsieh J, Schneider JW (2008) Cardiogenic small molecules that enhance myocardial repair by stem cells. Proc. Natl. Acad. Sci. USA 105:6063–6068.
17. Wu X, Ding S, Ding Q, Gray NS, Schultz PG (2004) Small molecules that induce cardiomyogenesis in embryonic stem cells. J. Am. Chem. Soc. 126:1590–1591.
18. Brand T (2003) Heart development: molecular insights into cardiac specification and early morphogenesis. Dev Biol 258: 1-19.
19. Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, Meloche S, et al. (2007) FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 134: 2895-2902.
20. Marguerie A, Bajolle F, Zaffran S, Brown NA, Dickson C, et al. (2006) Congenital heart defects in Fgfr2-IIIb and Fgf10 mutant mice. Cardiovasc Res 71: 50-60.
21. Wu X, Shi T, Ding S (2008) A chemical approach to stem-cell biology and regenerative medicine. Nature 453:338–344.
22. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448: 313-317.
23. Hsieh PCH, Davis ME, Gannon J, MacGillivray C, Lee RT (2006) Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. J Clin Invest 116: 237-248.
24. Tran TH, Wang X, Browne C, Zhang Y, Schinke M, et al. (2009) Wnt3a-induced mesoderm formation and cardiomyogenesis in human embryonic stem cells. Stem Cells 27: 1869-1878.
25. Itoh N, Ornitz DM (2011) Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. Journal of Biochemistry 149: 121-130.
26. Beenken A, Mohammadi M (2009) The FGF family: biology, pathophysiology and therapy. Nat Rev Drug Discov 8: 235-253.
27. Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, et al. (2006) Receptor specificity of the fibroblast growth factor family: the complete mammalian FGF family. J Biol Chem. 281: 15694–15700.
28. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, et al. (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448: 318-324.
29. Mauritz C, Schwanke K, Reppel M, Neef S, Katsirntaki K, et al. (2008) Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation 118: 507-517.
30. Christoforou N, Miller RA, Hill CM, Jie CC, McCallion AS, et al. (2008) Mouse ES cell–derived cardiac precursor cells are multipotent and facilitate identification of novel cardiac genes. J Clin Invest 118: 894-903.
31. Wu SM, Fujiwara Y, Cibulsky SM, Clapham DE, Lien C, et al. (2006) Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell 127: 1137-1150.
32. Kwon C, Qian L, Cheng P, Nigam V, Arnold J, et al. (2009) A regulatory pathway involving Notch1/β-catenin/Isl1 determines cardiac progenitor cell fate. Nat Cell Biol 11: 951-957.
33. Dell'Era P, Ronca R, Coco L, Nicoli S, Metra M, et al. (2003) Fibroblast growth factor receptor-1 is essential for in vitro cardiomyocyte development. Circ Res 93: 414-420.
34. Zhang J, Wilson GF, Soerens AG, Koonce CH, Yu J, et al. (2009) Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res 104: e30-41.
35. Martinez-Fernandez A, Nelson TJ, Yamada S, Reyes S, Alekseev AE, et al. (2009) iPS programmed without c-MYC yield proficient cardiogenesis for functional heart chimerism. Circ Res 105: 648-656.
36. Narazaki G, Uosaki H, Teranishi M, Okita K, Kim B, et al. (2008) Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells. Circulation 118: 498-506.
37. King T, Beddington RSP, Brown NA (1998) The role of the brachyury gene in heart
development and left–right specification in the mouse. Mech Dev 79: 29-37.
38. Saga Y, Miyagawa-Tomita S, Takagi A, Kitajima S, Miyazaki JI, et al. (1999) MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development 126: 3437-3447.
39. Conway SJ (1999) Novel expression of the goosecoid transcription factor in the embryonic mouse heart. Mech Dev 81: 187–191.
40. Nakashima Y, Ono K, Yoshida Y, Kojima Y, Kita T, et al. (2009) The search for Nkx2.5-regulated genes using purified embryonic stem cell-derived cardiomyocytes with Nkx2.5 gene targeting. Biochem Biophys Res Commun 390: 821–826.
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