系統識別號 U0026-3004201814421900
論文名稱(中文) 急性壓力造成憂鬱行為經由表觀遺傳學在海馬迴調控腦滋養因子
論文名稱(英文) Acute stress induced depressive-like phenotypes via epigenetic regulation of BDNF expression in hippocampus
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 106
學期 2
出版年 107
研究生(中文) 王品涵
研究生(英文) Pin-Han Wang
學號 S26054104
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 口試委員-張雋曦
中文關鍵字 憂鬱症  表觀遺傳學  神經滋養因子 
英文關鍵字 Major depressive disorder (MDD)  epigenetics  BDNF 
中文摘要 憂鬱症在全世界被視為一影響層面最深的精神疾患,歸因於現今治療上的侷限性,我們希冀以表觀遺傳學的角度切入,能在未來發展具前瞻性的藥物,以求臨床上更優越的療效。從病態生理學的角度探討憂鬱症之起因,降低神經滋養因子表現的推論已於先前廣泛被研究,但以表觀遺傳學之姿詮釋這現象,迄今尚未明確。我們以學習無助的憂鬱症動物模型,探討其憂鬱傾向的行為表現,藉由不同的實驗來確立此模型的效能,並將小鼠區分為兩類:憂鬱傾向與無憂鬱傾向。首先,從表觀遺傳學的組蛋白修飾談起,我們確立了組蛋白去乙醯化酵素二、五的表現與神經滋養因子變化的關聯性-增加組蛋白去乙醯化酵素二、五的表現量伴隨神經滋養因子下降。以小鼠腹腔注射丙戊酸,可觀察憂鬱傾向行為的改善及回升的神經滋養因子表現。第二,另一表觀遺傳學含攝的部分為基因的過度甲基化,甲基化的表徵顯現在蛋白質轉錄第一、三、及四的區域,此現象負向調節了神經滋養因子的表現。基因甲基化的重要媒介為基因甲基化轉移酶1、3a、3b。在憂鬱症傾向的小鼠顯現以下重要表徵-增強基因甲基化轉移酶1及3a的表現及降低腦滋養因子表現。我們藉此利用5-azacytidine(嘧啶核苷酸類似物,為一強效的甲基轉移酶抑制劑)反轉憂鬱傾向行為及改善神經滋養因子的表現。第三,利用體外實驗證實,微小核糖核苷酸能調控神經滋養因子三端非轉譯區的活性,miR-206-3p 能以互補鹼基配對的方式結合神經滋養因子三端的特定區域,以調節神經滋養因子的表現,遂被將此區視為微小核糖核苷酸的標的位點。共轉染微小核糖核苷酸的類似物或抑制劑,在冷光報導基因試驗及西方墨漬法中,均可見其劑量依賴的關聯性。我們分別在小鼠的海馬迴腦區及鼻腔給藥Cy3-AM206(即帶有Cy3螢光的miR-206-3p的修飾抑制劑),可清楚辨識Cy3螢光在腦中的分布,藉此以確認此藥投予的正確性,由降低非掙扎的時間及上升腦滋養因子的表現,斟酌神經滋養因子在行為及分子學上的效益。神經突觸在形態上的變化,以及相關突觸傳遞指標的螢光匯聚影像,均可作為AM206在療效上佐證的依據。結語:神經滋養因子被表觀遺傳學動態地調控,可被視為一個在臨床前期具治療潛能的藥物。
英文摘要 Major depression has been the most affected neuropsychiatric disorders in the world. We focused on the epigenetic mechanisms for developing potential antidepressants in the future. The reduction of BDNF expression in the pathophysiology of depressive-like behaviors has been extensively studied, but the role of epigenetic regulation in major depression remained elusive. We used learned helplessness animal model as the acute stressor and evaluated the depressive-like behaviors through different paradigms. We compared learned-helplessness (LH) mice with learned-helplessness-resilient (LHR) groups in a series of experiments. Firstly, we observed increased expression of HDAC2 and HDAC5 along with decreased BDNF in depressive state of rodents. Sodium valproate administrated in mice improved the depressive-like behaviors through enhancing BDNF. Secondly, there was significant increase of methylation on exon I, III , and IV, which negatively correlated with decreased BDNF. Then, we further surveyed the key methylation factors- DNA methyltransferase 1 , 3a ,and 3b(DNMT1,3a,3b). In LH mice, mRNA and protein level of DNMT1 and DNMT3a was elevated accompanied by the reduction of BDNF. Therefore, the potent DNMT inhibitor 5-azacytidine was used to evaluate the reversal of depressive-like behaviors and reduced BDNF expression. Thirdly, the modulation of BDNF 3’ UTR by miR-206 was carried out in vitro. The data indicated that miR-206-3p preferentially bind to the vectors bearing mBDNF #3, suggesting a targeted position for interfering BDNF expression. Cotransfection of mimics and inhibitors in vectors carrying mBDNF #3 displayed dose-dependent effects both in reporter assay and in BDNF expression. In vivo experiments, Cy3-AM206 was administrated in mice to verify the distribution of Cy3 fluorescence and beneficial effects on BDNF. Both intranasal and intra-hippocampal injection of AM206 in mice exhibited a therapeutic effect through behavior and molecular modulation, which was determined by decreased immobility time and enhanced BDNF. Morphological alteration of dendritic spines and colocalization of synaptic protein markers was also used to support the favorable effects of AM206.Taken together; BDNF expression is dynamically regulated by epigenetics and has been referred to a potential therapeutic agent in treating psychiatric disorders in preclinical study.
論文目次 Certificate for qualification
Abstract (in Chinese) I
Abstract (in English) IV
Acknowledgement VII
Tables of context IX
Figures and tables XII
Abbreviations XIII
Chapter 1: Introduction 1
1-1 Ideology 2
1-2 Current treatment for major depressive disorders 2
1-3 Hippocampus 2
1-4 Hippocampus and depression 2
1-5 BDNF genes 3
1-6 Structure of BDNF genes 3
1-7 BDNF expression in hippocampus 3
1-8 BDNF and depression 4
1-9 Epigenetics 4
1-10 Histone modifications 5
1-11 Histone modification in depression 5
1-12 Histone deacetylase inhibitors (HDACi) 5
1-13 DNA hyper-methylation 6
1-14 DNA hyper-methylation in depression 6
1-15 DNA hyper-methylation inhibitors and depression 7
1-16 MicroRNAs (miRNAs) 7
1-17 MicroRNAs (miRNAs) in depression 7
Chapter 2: Material and methods 12
2-1 Animals 13
2-2 Drugs 13
2-3 Learned-helplessness test (LHT) 13
2-4 Forced-swim test (FST) 14
2-5 Tail suspension test (TST) 14
2-6 Western blotting (WB) 14
2-7 Total RNA isolation 15
2-8 Real-time PCR of mRNA 16
2-9 DNA methylation assay 16
2-10 miRNA targets 17
2-11 Real-time PCR of miRNA 21
2-12 Dual luciferase assay 21
2-13 Treatment LH/LHR mice with antagomir 24
2-13-1Intra-hippocampal administration 24
2-13-2Intra-nasal administration 24
2-14 Analysis of dendritic spine density 25
2-15 Lentiviral transduction 25
2-16 Immunofluorescence 27
2-17 Statistical analyses 28
Chapter 3: Results 29
3-1 Depressive-like phenotypes are mediated by
epigenetic regulation of BDNF 30
3-2 Sod valproate reversed depressive-like phenotypes
by modulation of BDNF 31
3-3 The association of BDNF expression with
methylation pattern on individual variants 32
3-4 DNMTs enrichment negatively correlates with
relatively low expression of BDNF 33
3-5 5-azacytidine reverses the depressive-like
behaviors through regulation of BDNF 34
3-6 The suppressive effect of microRNAs (miR-206-
3p/5p) on BDNF 3’ UTRs in vitro 35
3-7 The beneficial effects of AM206 on BDNF in vivo 37
3-7-1 Intra-hippocampal administration 37
3-7-2 Intra-nasal administration 37
Chapter Four: Discussion 39
4.1 Overview of this study 40
4.2 BDNF expression and promoter methylation pattern 40
4.3 BDNF and miRNA 40
4.4 Animal model and disease 41
4.5 Conclusion 42
Chapter Five: Figures & Figure legends 43
Chapter Six: References 61
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