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系統識別號 U0026-3101201912463500
論文名稱(中文) 探討人類第九號纖維母細胞生長因子在亨丁頓氏手舞足蹈症中的保護與調節機制
論文名稱(英文) Investigating the protective roles and regulatory mechanisms of Fibroblast Growth Factor 9 in Huntington’s disease
校院名稱 成功大學
系所名稱(中) 跨領域神經科學國際博士學位學程
系所名稱(英) TIGP on The Interdisciplinary Neuroscience
學年度 107
學期 1
出版年 108
研究生(中文) 伊薩
研究生(英文) ISSA YUSUF
學號 SA8047012
學位類別 博士
語文別 英文
論文頁數 184頁
口試委員 指導教授-楊尚訓
共同指導教授-陳儀莊
召集委員-黃步敏
口試委員-莊季瑛
口試委員-孫孝芳
口試委員-吳佳慶
口試委員-莊佩錦
中文關鍵字 none 
英文關鍵字 Huntington’s disease  FGF9  apoptosis  oxidative stress  neuronal morphology  synaptic proteins 
學科別分類
中文摘要 none
英文摘要 Huntington’s disease (HD) is a heritable neurodegenerative disorder characterized by selective and progressive damage of medium spiny neurons in the striatum; and there is no cure for HD to date. A type of fibroblast growth factor (FGF), FGF9, has been reported to play prosurvival roles in other neurodegenerative diseases, such as Parkinson’s disease (PD), and Alzheimer’s disease (AD). With many similarities in the cellular and pathological mechanisms that eventually cause cell death in neurodegenerative diseases, we hypothesize that FGF9 might provide neuroprotective functions in HD. In this study, STHdhQ7/Q7 (WT) and STHdhQ111/Q111 (HD) striatal knock-in cell lines, and R6/2 primary cortical neurons were used to examine the neuroprotective effects and mechanisms of action of FGF9 on cell death, oxidative stress, neuronal morphology, and synaptic integrity in HD. Results show that FGF9 not only enhances cell proliferation, but also suppresses cell death, alleviates oxidative stress, enhances neuronal morphology, and increases synaptic protein expressions. FGF9 significantly upregulates glial cell line-derived neurotrophic factor (GDNF), upregulates an anti-apoptotic protein, Bcl-xL, decreases the expression of an apoptotic protein Bax, and suppresses apoptotic marker, cleaved caspase 3; mostly through activating NF-kB pathway in HD cells. In addition, FGF9 upregulates Nrf2 expression and activation, increases Nrf2-ARE activity, and subsequently upregulating downstream targets of Nrf2 including SOD2, GCLc, and GR. Furthermore, FGF9 increases neurite outgrowth and neuron morphology proteins including βIII-tubulin, MAP2A/B, and GAP-43 protein expressions; and upregulates the expression of synaptic proteins including Synaptophysin and PSD-95, mostly through activating NF-kB pathway in HD cells. FGF9 may function through ERK, AKT and JNK pathways. Especially, ERK pathway plays a critical role to influence the effects of FGF9 on cell death, oxidative stress, neuronal morphology and synaptic integrity. The results obtained in this study not only show the neuroprotective effects of FGF9, but more importantly, clarify some critical mechanisms in HD cells, further providing an insight for the therapeutic potential of FGF9 in HD.
論文目次 Table of Contents
Abstract i
Acknowledgement iii
Figure contents ix
Abbreviations xi
1 Chapter 1 General Introduction 1
1.1 Huntington’s disease 1
1.2 Fibroblast growth factors (FGFs) 3
1.2.1 Fibroblast growth factors signalling 4
1.2.2 Fibroblast growth factors in Huntington’s diseases 8
1.2.3 ERK signalling pathway in Huntington’s disease 9
1.3 Significance of study 11
1.4 Aim and Hypothesis of study 12
2 Chapter 2 Investigating the pro-survival role of FGF9 in Huntington’s disease 13
2.1 Introduction 14
2.1.1 Apoptosis 14
2.1.2 Apoptosis in Huntington’s disease 14
2.1.3 Neurotrophic factor therapy in Huntington’s disease 16
2.1.4 NF-kB: A regulator of cell survival 17
2.1.5 Pro-survival role of Fibroblast growth factors 19
2.2 Aim and Hypothesis 19
2.3 Materials and Methods 21
2.3.1 Cell culture and treatments 21
2.3.2 Subculture of cells 21
2.3.3 Freezing of cells 22
2.3.4 Thawing out of cells 22
2.3.5 Primary cortical neuron culture 23
2.3.6 Genotyping for R6/2 transgenic mice 23
2.3.7 Western blotting 24
2.3.8 DNA construction 27
2.3.9 Plasmid DNA extraction 27
2.3.10 Luciferase reporter assay 28
2.3.11 Immunofluorescence staining 29
2.3.12 Propidium iodide (PI) staining 29
2.3.13 MTT Assay 30
2.3.14 MAP2 staining for neuron survival 30
2.3.15 Statistical analysis 30
2.4 Results 32
2.4.1 Huntington’s disease cells are vulnerable to serum starvation 32
2.4.2 FGF9 induces cell proliferation in HD 33
2.4.3 FGF9 plays protective roles against cell death in HD cells 33
2.4.4 FGF9 upregulates GDNF protein expression in HD cells 34
2.4.5 FGF9 upregulates and activates NF-kB in HD cells 35
2.4.6 NF-kB mediate the pro-survival role of FGF9 in Q111 cells 36
2.4.7 FGF9 activates AKT, ERK, and JNK signalling pathways 37
2.4.8 FGF9 pro-survival role in HD cells is mediated through ERK signalling pathway 38
2.5 Discussion 40
2.6 Figures 46
3 Chapter 3 Investigating the role of FGF9 on Oxidative stress in Huntington’s disease 77
3.1 Introduction 78
3.1.1 Oxidative stress 78
3.1.2 Oxidative stress in Huntington’s disease 79
3.1.3 Antioxidant therapy in Huntington’s disease 80
3.1.4 Nrf2-ARE pathway: A master regulator of antioxidant response 81
3.1.5 Fibroblast growth factors in oxidative stress 83
3.2 Aim and hypothesis 84
3.3 Materials and Methods 85
3.3.1 Cell culture and treatments: 85
3.3.2 DNA construction 85
3.3.3 Western blotting: 85
3.3.4 Nuclear and cytoplasmic protein extraction 85
3.3.5 Luciferase reporter assay 86
3.3.6 Knockdown of Nrf2 86
3.3.7 Dihydroethidium (DHE) assay 87
3.3.8 Statistical analysis 87
3.4 Results 88
3.4.1 Serum starvation induces oxidative stress in HD cells 88
3.4.2 FGF9 protects against oxidative stress in HD cells 88
3.4.3 Serum starvation impairs Nrf2 response in HD cells 89
3.4.4 FGF9 promotes Nrf2 upregulation and activation in HD cells 89
3.4.5 FGF9 increases Nrf2-ARE activity 90
3.4.6 Serum starvation impairs Nrf2 downstream antioxidant targets in HD cells 90
3.4.7 FGF9 upregulates Nrf2 downstream antioxidant targets in HD cells 92
3.4.8 Nrf2 mediates FGF9 protective effects against oxidative stress in HD cells 92
3.4.9 FGF9 effect against oxidative stress in HD cells is mediated in part through ERK signalling pathway 93
3.5 Discussion 95
3.6 Figures 100
4 Chapter 4 Investigating the role of FGF9 on neuronal morphology and synaptic integrity in Huntington’s disease 129
4.1 Introduction 130
4.1.1 Neuronal morphology 130
4.1.2 Synaptic formation and plasticity 131
4.1.3 Neuronal morphology in Huntington’s disease 132
4.1.4 Synaptic functions in Huntington’s disease 133
4.1.5 NF-kB in neuronal morphology, synaptic formation and plasticity 135
4.1.6 Fibroblast growth factors in neuronal morphology, synaptic formation and plasticity 136
4.2 Aim and hypothesis 137
4.3 Materials and Methods 139
4.3.1 Cell culture and treatments 139
4.3.2 Western blotting 139
4.3.3 Immunofluorescence staining 139
4.3.4 Statistical analysis 140
4.4 Results 141
4.4.1 FGF9 increases neuronal outgrowth in HD cells 141
4.4.2 FGF9 upregulates neuronal morphology related proteins in HD cells 141
4.4.3 FGF9 upregulates synaptic proteins in HD cells 142
4.4.4 FGF9 regulates neuronal morphology and synaptic proteins through NF-kB pathway 143
4.4.5 FGF9 enhances neuronal morphology, and synaptic formation through ERK signalling pathway in HD cells 143
4.5 Discussion 145
4.6 Figures 151
Chapter 5 Conclusions 163
Chapter 6 Reference 167

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