博碩士論文 105821608 詳細資訊




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姓名 司峇塔(BASTA SIMANULLANG)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 在空間學習和記憶形成後對海馬迴中表觀遺傳修飾調控 miRNA 表現的研究研
(The investigation of the epigenetic modification regulated mi-RNA induction in the hippocampus after spatial learning and memory formation)
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摘要(中) 行為是由環境刺激驅動的,這種刺激通過許多分子機制觸發大腦活動。學習和記憶的過程包括
刺激誘導的海馬神經元之間的突觸連接的變化,其在長期的體細胞和情節記憶的形成中起關鍵作用。通
過Morris 水迷宮實驗與RNA 的分析表明,在有學習和未學習的小鼠之間存在微小RNA 表達不同。屬
於轉錄後調節因子的miR-466f-3p 在腦中大量表現且其在基因中位於一基因的內含子-10 中(Sfmbt2)。然
而,宿主基因(sfmbt2)的mRNA 表現量與miR-466f-3p 顯著不同。表觀遺傳修飾,如DNA 甲基化可影
響基因表達。5′Aza-dC,一個甲基轉移酶抑製劑,用於處理海馬迴原代培養神經元導致Bdnf,Nrf2 和Sfmbt2
mRNA 與miR466f-3p 的表現量增加。根據實驗結果顯示,miR-466f 家族的啟動子區域中其甲基化模式
在有學習和未學習的小鼠之間沒有顯著差異。證據表明,在Morris 水迷宮的應激下,DNA 甲基化的變化
不直接參與誘導miR-466f-3p 在海馬依賴性空間學習和記憶形成的調節。
摘要(英) Behavior is driven by environmental stimulation that triggers brain activity with lots of
molecular mechanisms. The process of learning and memory contains stimulation-induced
changes that happen in the synaptic connections between neurons in the hippocampus, which
plays a pivotal role in the formation of long term somatic and episodic memories. By Morris
water maze experiment and RNA analysis, we show that some micro-RNAs (miRNA) are
expressed differently between learned and unlearned mice. One of the miRNAs, miR-466f-3p,
which belongs to post transcriptional regulator, is abundantly express in brain and generated in
the intron-10 of Sfmbt2 gene. However, the mRNA expression of Sfmbt2 is significantly
different from miR-466f-3p. Inhibition of epigenetic modification, such as DNA methylation,
can affect gene expression. Treatment of 5’Aza-dC, a methyltransferase inhibitor, to the
hippocampal primary culture neuron causes Bdnf, Nrf2, Sfmbt2 mRNA and miR466f-3p
expression increased. However, the methylation pattern in the promoter region of the miR-466f
cluster is not significantly different between learned and unlearned mice. The evidence show
that the changes of DNA methylation under the stress of the Morris water maze indirectly
participate in the regulating of miR-466f-3p induction in the hippocampus-dependent spatial
learning and memory formation.
Keywords: Epigenetic, mi-RNAs induction, Methylation
關鍵字(中) ★ 表觀遺傳
★ mi-RNAs 誘導
★ 甲基化
關鍵字(英) ★ Epigenetic
★ mi-RNAs induction
★ Methylation
論文目次 Table of Contents
Abstract . i
中文摘要 ii
Acknowledgement iii
Table of Contents . iv
List of Figures . vi
List of Tables . vii
Abbreviations .. viii
Chapter 1 Introduction . 1
1.1. Learning and Memory . 1
1.2. Sfmbt2 host gene, miRNA cluster and memory formation .. 2
1.3. MicroRNA biogenesis . 2
1.4. Epigenetic modification .. 2
1.4.1. Methylation 3
1.4.2. 5’Aza-dC as a methytransferase inhibitor 3
1.4.3. Sodium Bisulfate Treatment 4
1.5. Research and Purposes 4
Chapter 2 Materials and Methods .. 6
2.1. Morris water maze task 6
2.2. Genomic DNA extraction .. 6
2.3. Bisulfate treatment . 6
2.4. Epi-tag PCR .. 7
2.5. Gel-PCR purification 8
2.6. Blunting and ligation 8
2.7. Transformation . 8
2.8. Colonies screening . 9
2.9. DNA mini preparation . 9
2.10. Sequencing analysis 10
2.11. Cell line culture . 10
2.12. Hippocampal primary culture 10
2.13. RNA purification . 11
2.14. Reverse transcriptase PCR (RT-PCR) 11
2.15. Polymerase chain reaction (PCR) 12
2.16. Quantitative polymerase chain reaction (Q-PCR) .. 12
2.17. MicroRNA RT-Q-PCR .. 13
Chapter 3 Results . 14
3.1. mRNA expression of Sfmbt2 is increase in N2a cell line under 5’Aza-dC treatment at a time-and dose-dependent manner 14
3.2. Sfmbt2 mRNA expression is increased in primary hippocampal neuron under 5’Aza-dC treatment in a time and dose dependent manner 14
3.3. Percentage of DNA methylation in some specific CpG sites are significantly different before and after training of C57BL/6J Wild-Type (WT) mice .. 15
3.4. DNA Methylation of F1 generation is hypomethylated compare to F0 in hippocampus.. 15
3.5. DNA methylation pattern in hippocampus of F1 mice before and after training 16
3.6. DNA methylation pattern in liver of F0 and F1 (LNxWT) mice before and after training .. 17
Chapter 4 Discussion . 18
Chapter 5 Conclusion 21
References .. 22
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指導教授 沈哲鯤(Dr. Che-Kun James Shen) 審核日期 2019-8-1
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