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研究生: 林宏諭
Lin, Hung-Yu
論文名稱: FGF1B啟動子之活化及FGF1訊息傳遞於心臟生成分化過程所扮演的角色
FGF1B Promoter Activation and FGF1 Signaling in Cardiogenesis
指導教授: 邱英明
Chiu, Ing-Ming
汪宏達
Wang, Horng-Dar
口試委員: 喻秋華
紀雅惠
許益超
學位類別: 博士
Doctor
系所名稱: 生命科學暨醫學院 - 生物科技研究所
Biotechnology
論文出版年: 2016
畢業學年度: 104
語文別: 英文
論文頁數: 94
中文關鍵詞: FGF1B啟動子, FGF1, 心臟生成
外文關鍵詞: FGF1B, FGF1, cardiogenesis
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  • 中文摘要
      心血管疾病,於廿一世紀人類十大死因排行榜上,排名第一位。對於治療心血管疾病,心臟移植治療長久以來都被視為是可行的療法,但是,適宜的捐贈者來源太過於稀少,而無法用以廣泛的醫治病患;因此,細胞移植療法被視作一種替代心臟移植治療的可行性方案,而被廣泛的研究。故,能促進心臟生成的基因或因子,也會被認定為有潛能用以治療心血管疾病。FGF1,於先前的文獻報導中,已被發現在心肌梗塞實驗狀態下,能促進心肌再生;然而,FGF1如何誘導心肌再生的詳細機制,仍不知曉。在這篇研究中,我們利用化學藥物誘導老鼠胚胎幹細胞進行細胞分化,建立了體外心臟生成分化的模式;用以研究FGF1及其調控因子們,在心臟生成的狀態下所扮演的功能角色。催產激素,除了是一耳熟能詳的賀爾蒙,更是一個可靠的體外誘導心臟生成分化之化學物品;藉由其誘導,我們成功的在老鼠胚胎幹細胞株E14Tg2a上建立體外心臟生成分化模式。藉由心臟生成分化步驟,於分化後期(分化第八至十四天)可以觀察到自律性跳動的心肌細胞簇;於此同時,FGF1的調控因子,FGF1B啟動子,其信使核醣核酸表現量亦會大幅度地上升。引人注目的是,FGF1B啟動子在心臟生成分化時期的信使核醣核酸表現量圖形和成熟心肌之特徵基因,心肌肌鈣蛋白T的信使核醣核酸表現量圖形相仿。抑制FGF1及FGFR的訊息傳遞,則是會造成自律性跳動的心肌細胞簇的減少,也會導致成熟心肌分化所需相關基因的信使核醣核酸表現量下降。此外,藉由篩選FGF1的下游訊息傳遞鏈,我們發現到,控制自律性跳動的心肌細胞簇之形成,主要是透過FGF1-FGFR-PKC的訊息傳遞來調控。整合以上發現,我們的研究提供了證據,支持FGF1確實可調控心臟生成的分化過程;並且,FGF1主要藉由FGF1-FGFR-PKC此訊息傳遞鏈來協調心臟生成分化過程,而非透過FGF1-FGFR-MAPK此另一訊息傳遞鏈來調控。


    Abstract
      Heart disease is the leading cause of human death in the 21st century. Heart transplantation is a promising way to treat this. Because donor resources are limited, cell-based therapy has been developed as an alternative. Therefore, genes that trigger cardiogenesis could have potential in the treatment of heart disease. FGF1 is reported to stimulate cardiomyocyte proliferation under conditions of myocardial infarction; but little is known about its function during cardiac differentiation. In this study, we established an in vitro cardiogenesis model through a reliable chemical induction protocol to determine whether FGF1 and its gene expression are involved in cardiogenesis. Oxytocin, a well-known hormone but also a cardiac differentiation inducer, was used in a mouse embryonic stem cell line E14Tg2a to achieve cardiac differentiation. After differentiation, beating cell clusters appeared and the expression of FGF1B mRNA was upregulated in the late differentiation stage (differentiation days 8–14). Interestingly, FGF1B expression patterns during cardiac differentiation were similar to those of a mature cardiomyocyte marker, troponin T2, cardiac. The blockage of FGF1-FGFR signaling reduced not only the appearance of beating cluster formation but also the expression levels of cardiomyocyte-associated genes. Moreover, by investigating FGF1 downstream signaling cascades, we observed that the efficiency of beating cluster formation was mainly regulated via the FGF1-FGFR-PKC signaling axis. Taken together, we provide evidence to support that FGF1 could regulate cardiogenesis primarily through the PKC signaling, but not through the MAPK signaling pathway.

    Table of contents 中文摘要 ⅰ 英文摘要 ⅲ 致謝 ⅳ List of Abbreviations ⅴ I: Introduction 1   1.1 Cardiovascular disease 1     1.1.1 Cardiovascular disease in worldwide and in Taiwan 1     1.1.2 Cardiac disease therapies 1   1.2 Heart regeneration 2     1.2.1 Heart regeneration in amphibian and zebrafish 2            1.2.2 Heart regeneration in mammals 4   1.3 Stem cells and cell therapy in heart 5     1.3.1 Cardiac stem/progenitor cells in heart 5     1.3.2 In vitro induction of cardiogenesis using embryonic stem cells 6     1.3.3 Stem/progenitor cells in cardiovascular disease therapies 7   1.4 Fibroblast growth factors 10     1.4.1 Members of fibroblast growth factor family 10     1.4.2 Fibroblast growth factor signaling pathways 11     1.4.3 Fibroblast growth factor 1 11     1.4.4 Fibroblast growth factors in cardiogenesis 12   1.5 The aims of this study 14 II: Materials and Methods 15   2.1 Zebrafish 15     2.1.1 Zebrafish husbandry and embryo microinjection 15     2.1.2 Zebrafish heart cell culture and transfection of human FGF1B-540-GFP       plasmid DNA 15   2.2 Mouse 16     2.2.1 Culture of mouse heart primary cells and transfection of human FGF1B        -540-GFP plasmid DNA 16     2.2.2 Culture of mouse embryonic stem cells 17     2.2.3 In vitro cardiac differentiation 17     2.2.4 RNA preparation, reverse transcription-polymerase chain reaction,        and real-time polymerase chain reaction 18     2.2.5 Immunostaining, Western blotting and fluorescence activated cell sorting      (FACS) 19     2.2.6 Blockage of FGF1 and its downstream signaling cascades 21   2.3 Statistical analyses 22      III: Results 23   3.1 Cardiogenesis in zebrafish 23     3.1.1 Human FGF1B-540-GFP plasmid DNA could be expressed in zebrafish heart region in vivo 23     3.1.2 Human FGF1B-540-GFP plasmid DNA was expressed in zebrafish heart cells after electroporation 23     3.1.3 The FGF1 amino acid sequence homology data revealed Zebrafish is not       an appropriate model to investigate the function of FGF1 in cardiogenesis 24        3.2 Cardiogenesis in mouse 25     3.2.1 Human FGF1B-540-GFP could be activated in cultured mouse heart cells 25     3.2.2 Oxytocin treatment of mouse ES cells resulted in cardiac differentiation 26     3.2.3 Immunostaining analysis, Western blotting and FACS revealed terminal       cardiac differentiation 27     3.2.4 FGF1B mRNA levels increased during cardiac differentiation 28     3.2.5 Blockage of FGF1 signaling decreased the efficiency of beating cluster         formation and reduced the expression levels of cardiomyocyte marker        genes 29     3.2.6 FGFR inhibitor reduced the efficiency of beating cluster formation and the         mRNA levels of cardiomyocyte marker genes, and the reduction was rescued       by PMA 30     3.2.7 Protein kinase C was the main regulator downstream of FGF1-FGFR       to mediate beating cluster formation 31 IV: Discussion 33   4.1 The function of FGF1B promoter could be conserved in human and in zebrafish 33   4.2 The link between oxytocin and FGF signaling 34   4.3 The FGF1A mRNA expression levels maintain in consistent levels during cardiac differentiation 34   4.4 The possible role of FGF1A in cardiogenesis 35   4.5 FGF1 could stimulate angiogenesis and cardiomyogenesis 36   4.6 Downstream of FGF1 mainly through FGFR-PKC signaling regulates cardiogenesis 36   4.7 FGF1 signaling pathways enhances cardiogenesis 37   4.8 Multiple signaling pathways downstream of FGF1 could regulate cardiogenesis     in the same differentiation stage 39   4.9 FGF1B transgenic mice could help to elucidate the cell resource responsible     for cardiac regeneration 40   4.10 The FGF1B positive and negative cells may have different functions in     cardiogenesis 40   4.11 Recent breakthrough investigations on cardiac regeneration 41   4.12 Basic researches combined new biomaterials give promising ways for exploring therapies for cardiovascular diseases 42 V: Conclusion 44 List of Figures and Figure Legends VI: Figures and Figure Legends 45   6.1 FIG. 1. Human FGF1B-540-GFP plasmid DNA could express in zebrafish    heart region in vivo. 45   6.2 FIG. 2. Human FGF1B-540-GFP plasmid DNA expressed in zebrafish heart cells    after electroporation. 47   6.3 FIG. 3. Amino acid sequence alignment of FGF1 and FGF1-like proteins among        different species. 49   6.4 FIG. 4. Amino acid sequence alignment of FGF2 proteins among different species. 51   6.5 FIG. 5. Human FGF1B-540-GFP could be activated in cultured mouse heart cells. 53   6.6 FIG. 6. Oxytocin treatment of mouse ES cells resulted in cardiac differentiation. 55   6.7 FIG. 7. Immunostaining analysis, Western blotting and FACS revealed terminal     cardiac differentiation. 57   6.8 FIG. 8. FGF1B mRNA levels increased during cardiac differentiation. 59   6.9 FIG. 9. Blockage of FGF1 signaling decreased the efficiency of beating cluster     formation and reduced the expression levels of cardiomyocyte marker genes. 61   6.10 FIG. 10. FGFR inhibitor reduced the efficiency of beating cluster formation     and the mRNA levels of cardiomyocyte marker genes, and the reduction was rescued by PMA. 63   6.11 FIG. 11. Protein kinase C was the main regulator downstream of FGF1-FGFR     to mediate beating cluster formation. 65   6.12 FIG. 12. Administration of FGF1 related inhibitors blocked FGF1 signaling. 67   6.13 FIG. 13. Treatments with FGF1 related inhibitors did not cause apparent toxic     effects on cells. 69   6.14 FIG. 14. FGF1-FGFR-PKC signaling axis regulates beating cluster formation     and cardiomyocyte differentiation in the late differentiation stage. 71    List of Tables VII: Tables 73   7.1 Table1: Comparison of the percent identities at the amino acid level among FGF1     and FGF1-like proteins from different species 73   7.2 Table2: Comparison of the percent identities at the amino acid level of the three     coding exons between human FGF1 and each of the two zebrafish FGF1-like     proteins 74   7.3 Table3: Comparison of the percent identities at the amino acid level among FGF2     from different species 75 List of Movies VIII: Movies 76   8.1 Movie1. PMA rescues SU5402 caused beating cell cluster formation lost effect 76   8.2 Movie2. PMA rescues STA caused beating cell cluster formation lost effect 77 IX: References 78 X: List of Publications 94

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