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研究生:安娜
研究生(外文):Endang Mariana Simamora
論文名稱:EPO細胞訊息系統在神經發育中的功能
論文名稱(外文):Functions of EPO Signaling in Embryonic Neurogenesis
指導教授:胡清華胡清華引用關係
指導教授(外文):Chin-Hwa Hu
口試委員:管永恕黃聲蘋鄒文雄胡清華
口試委員(外文):Yung-Shu KuanSheng-Ping L. HwangWen-Shyong TzouChin-Hwa Hu
口試日期:2020-01-15
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生命科學暨生物科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:48
中文關鍵詞:斑馬魚(Zebrafish)紅血球生成素(Erythropoietin)神經分化(Neuron Differentiation)
外文關鍵詞:ZebrafishErythropoietinNeuron Differentiation
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紅血球生成素(Erythropoietin, EPO)是一種醣蛋白激素,能控制紅血球前驅細胞的生存、增殖和分化。EPO最初是因為造血過程中起關鍵作用而得名,而後研究發現它在神經發育中也扮演了關鍵作用。EPO受體(EPO receptor, EPO-R)為第一類細胞激素受體中的一種,可藉由各種蛋白激酶和STAT轉錄因子來證明其作用。目前為止,有關EPO如何促進神經細胞生成的詳細機制尚不清楚。而在這項研究中,主要在探討Epo信號傳遞系統影響神經發育的機制。實驗中,利用morpholino敲落epo基因,會減少xiap / birc4和ciap1 / birc2的轉錄作用以及抑制了神經的分化。此外,敲落xiap / birc4和ciap1 / birc2也影響到神經發育,證實了XIAP和cIAP1在神經發育中扮演著相當重要的角色。實驗發現以AG490抑制Jak2或者敲落stat5基因皆可降低xiap / birc4和ciap1 / birc2的轉錄,抑制神經的生成。利用異位表達的xiap / birc4和ciap1 / birc2 mRNA顯微注射入胚胎中,可以復原敲落Epo或以AG490處理的胚胎內神經的分化。總而言之,此項研究表明Epo信號傳遞會藉由激活Jak2 / STAT5並上調xiap / birc4和ciap1 / birc2轉錄來調節胚胎神經的發育。
Erythropoietin (EPO) is a glycoprotein cytokine required for the survival, proliferation and differentiation of the erythrocytic progenitors. Erythropoietin (EPO) was originally named because of their pivotal contribution to hematopoiesis, but it also plays critical roles in neurogenesis. EPO receptor (EPO-R) is a member of class I cytokine receptor family that excerts its effects through various protein kinases and STAT transcription factors. The mechanism how EPO modulates neurogenesis still remains to be clarified. In this study, I would like to investigate how Epo signaling mediate neurogenesis. It shows that morpholino mediated knockdown of epo results in decrease of xiap/birc4 and ciap1/birc2 transcription and inhibition of neuronal differentiation. Furthermore, knockdown of xiap/birc4 and ciap1/birc2 also abrogates neural development, suggesting that XIAP and cIAP1 play important roles in neurogenesis. Inhibiting Jak2 activity with AG490 or knockdown of stat5 also blocks transcription of xiap/birc4 and ciap1/birc2 and inhibits neural development. Ectopic xiap/birc4 and ciap1/birc2 mRNA could rescue the neuronal differentiation in epo morphants and AG490-treated embryos. In summary, this study suggests that Epo signaling modulates embryonic neurogenesis by activating Jak2/Stat5 and upregulating xiap/birc4 and ciap1/birc2 transcriptions.
摘要 i
ABSTRACT ii
TABLE OF CONTENTS iii
FIGURE INDEX v
CHAPTER I INTRODUCTION 1
1.1. Erythropoietin (EPO) 1
1.2. JAK2 / STAT5 1
1.3. Inhibitor of Apoptosis (IAPs Family) 3
1.4. Expression of Neuro-D (nrd) and elav (HuC) during neuronal differentiation 3
CHAPTER II MATERIALS AND METHODS 5
2.1. Materials 5
2.1.1. Model Animal 5
2.1.2. Competent cells and Plasmids 5
2.1.3. Chemicals used for experiments 5
2.1.4. Enzymes and commercial kits 5
2.1.5. Experimental Chemical Preparation 5
2.2. Methods 6
2.2.1. Collect zebrafish embryos and treatment drugs 6
2.2.2. Morpholino injection 7
2.2.3. Ribonucleic Acid Extraction (Total RNA Extraction) 7
2.2.4. cDNA preparation 8
2.2.5. Polymerase Chain Reaction (PCR) 8
2.2.6. DNA Ligation 8
2.2.7. Transformation 8
2.2.8. Plasmid Extraction (Miniprep kit) 9
2.2.9. DNA Amplification by PCR 9
2.2.10. Making RNA Probe 9
2.2.11. Whole mount in situ Hybridization 10
2.2.12. Quantitative RT-PCR (qPCR) 11
2.2.13. mRNA Rescue Essay 12
2.2.14. Polyadenylation 13
CHAPTER III RESULTS 14
3.1. Transcriptions of XIAP and cIAP1 are controlled by Epo-Jak2 signaling 14
3.2. Jak2/Stat5-IAPs signaling pathway protects neural cells against apoptosis 14
3.3. Epo-Jak2/Stat5-IAPs signaling pathway is important for neural differentiation 15
CHAPTER IV DISCUSSION 16
REFERENCES 17
FIGURES SECTION 20
APPENDIX 37
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