臺灣博碩士論文加值系統

微型核醣核酸（microRNA）是一段長約22個核苷酸的核醣核酸（RNA），在細胞內扮演轉譯後調控的角色。微型核醣核酸在大腦中參與許多的分子調節機制，例如：樹突的形成、突觸的組成、突觸的成熟，這些現象都與神經可塑性相關。先前研究指出體外培養的海馬神經細胞中微型核醣核酸132（miR-132）會促進神經樹突的生長以及樹狀突脊的形成，這些神經型態的改變與記憶的形成有很大的相關性。miR-132其下游基因包含p250 GTPase-active protein （p250GAP）和methyl CpG binding protein 2（MeCP2），藉由miR-132的調控可能影響到大腦中的記憶功能，先前miR-132與神經塑型及學習記憶相關研究皆為體外細胞培養實驗，缺乏活體動物及其行為的實驗證據，因此本論文利用恐懼制約訓練，研究小鼠miR-132的表達量對動物恐懼記憶的影響。本論文研究結果顯示：以慢病毒攜帶miR-132拮抗子送入海馬後可成功抑制大腦海馬miR-132的表達約20%，並使小鼠痕跡恐懼制約的習得能力下降，但抑制miR-132並影響習得的過程並未改變p250GAP、CREB的表達，而MeCP2的表達比ACSF組別低30%。此外，小鼠痕跡恐懼制約情境測試的提取促進左海馬miR-132的表達較右邊高出40％，顯示訓練後miR-132在海馬內有不對稱表達性。且在右海馬內MeCP2蛋白的表達高對照組20%。若抑制miR-132後再提取情境記憶時發現海馬內p250GAP、MeCP2蛋白的表達增加，CREB蛋白則無變化。然而，抑制miR-132並未改變痕跡恐懼記憶的提取行為表現，推測提取後所觀察到miR-132表達的改變與記憶提取本身無關，而可能與提取後記憶重新統整（reconsolidation）的過程有關。

MicroRNAs (miRNAs) are small non-coding RNAs that play as post-transcriptional regulators in various cellular processes. MicroRNA-132 (miR-132) has been shown to affect dendritic growth and spinogenesis in cultured hippocampal neurons and in brain slices. It has been known that p250 GTPase-active protein (p250GAP) and methyl-CpG-binding protein 2 (MeCP2) translation is regulated by miR-132. However, its physiological effects in vivo and correlation with behavioral tasks remain unclear. Therefore, this thesis aims to investigate the role of miR-132 in the hippocampal region of mice after fear conditioning. Half an hour after trace fear conditioning, miR-132 expression level was increased in the bilateral hippocampi. Next, engineered miR-132 antagomer carried by lentivirus to was infused to the hippocampi to knockdown local miR-132 expression and the effect was correlated with fear learning behavior. MicroRNA-132 expression level was successfully knocked down by 20 %, which was associated with significant learning deficit in trace fear conditioning. Furthermore, level of p250GAP and cAMP response element binding protein (CREB) was not significantly different when miR-132 knockdown in fear learning behavior. Additionally, level of miR-132 of trace fear conditioning (TFC) group was significantly higher than Naïve group in the left hippocampus after retrieval of contextual fear memory and asymmetrical expression pattern of miR-132 was observed. Moreover, after retrieval of contextual fear memory knockdown of miR-132 the in hippocampus increased expression of MeCP2 and p250GAP, but did not change CREB. Knockdown of miR-132 did not change freezing behavior to context, suggesting that miR-132 expression may affect later processes related to memory consolidation instead of retrieval of contextual memory per se.

Abstract I
中文摘要 III
目錄 0
壹、 序論 1
1. 學習記憶的分子機轉 1
2. 恐懼制約模式 3
3. 微型核醣核酸與學習記憶 4
4. 微型核醣核酸132（miR-132） 7
5. 微型核醣核酸132與學習障礙及腦神經發育異常/退化性疾病 10
貳、 實驗目的 13
參、 實驗設計與流程 14
肆、 材料與方法 16
1. 實驗動物 16
2. 備製病毒 16
3. 降低miR-132表達量過程 20
4. 恐懼制約試驗（Fear conditioning test） 22
5. 灌流（Perfusion） 24
6. 冷凍切片（Frozen section） 25
7. 螢光顯微鏡 25
8. 萃取海馬的核醣核酸（RNA） 25
9. 即時定量微型核醣核酸 26
10. 海馬蛋白質的萃取 27
11. 西方墨點法（Western blot analysis） 28
12. 統計分析 31
伍、 實驗結果 32
陸、 討論 39
柒、 圖表 45
捌、 參考文獻 63

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