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研究生:陳怡婷
研究生(外文):Yi-Ting Chen
論文名稱:探討TDP基因轉殖鼠在神經細胞形態之研究
論文名稱(外文):The Study of Neuronal Morphology in TDP Transgenic Mice
指導教授:沈哲鯤沈哲鯤引用關係
指導教授(外文):Che-Kun James Shen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:59
中文關鍵詞:海馬迴學習/記憶能力微注射技術刺狀突起(突刺)神經纖維細胞本體
外文關鍵詞:TAR DNA binding protein-43 (TDP-43)fragile X mental retardation protein (FMRP)Staufen 1TDP-43-positive ubiquitinated inclusions (UBIs)hippocampuslearning/memory capabilitiessingle-cell microinjectiondendritic spines
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在過去,TAR DNA binding protein-43(TDP-43)是ㄧ個眾所周知的nuclear factor,擁有調控轉錄抑制和選擇性剪接進行的功用。直到近年來更發現TDP-43會協同FMRP和Staufen 1,這兩個已知能夠調控神經活性,和學習記憶形成建立有關的RNA結合蛋白。有趣的是,在許多神經退化性疾病案例中,包括額顳葉退化變性及肌萎縮性脊髓側索硬化症(漸凍人),TDP-43都會不正常的聚集堆積,亦即從原本正常表現在細胞核裡,會不正常的以inclusion body型式沉降堆積於患者大腦中神經細胞的細胞質中。
在我們實驗室裡,更發現TDP-43過度表達在腦部特定區域(海馬迴及大腦皮質)的基因轉殖鼠,在行為測試中,學習/記憶的能力相較於正常小鼠是表現較差的,故本論文在於探討細胞層面上,以微注射技術(microinjection)去比較正常小鼠和基因轉殖鼠在單一神經細胞形態及連結上的差異,以了解學習記憶的喪失,TDP-43是否扮演具有關鍵性的角色。在利用Sulforhodamine101和Biocytin這兩種不同染劑微量注射的結果,我們量化分析足以代表神經細胞在外觀形態上的特徵數個參數(parameters)並發現不論是總神經纖維長度、從細胞本體所生長出去的神經纖維主幹、神經纖維分支數及平均每根神經纖維主幹的長度,在不同時間點的量化下(體外培養7、14、19和21天)都沒有明顯的差異。然而,從Biocytin微量注射以及實驗室現有的GFP螢光小鼠和TDP基因轉殖鼠做交配(TDP+/- GFP+/-)所得到的初代海馬迴細胞培養(primary hippocampal culture),可以清楚地看到代表細胞連結的細微結構-刺狀突起(突刺)從體外培養第七天開始,TDP基因轉殖鼠的突刺密度都是較低的,和正常小鼠存在有統計意義上的差異。暗示並強調這些研究結果(突刺密度的降低)可能具有重要的涵義對於了解學習記憶的低下遲緩,都與TDP過度表現有關。
In the past, TAR DNA binding protein-43 (TDP-43) is a well-known nuclear factor that carried out dual functions, both as a transcriptional repressor and an activator of exon skipping, implicated in alternative splicing. Until a couple of years ago, it was found that TDP-43 colocalized with other two RNA-binding proteins, fragile X mental retardation protein (FMRP) and Staufen 1, which are known to regulate neuronal activities, involving in the establishment of learning/memory capabilities. Interestingly, TDP-43-containing inclusions bodies aggregate in the cytosol in many neurodegenerative diseases, featuring TDP-43-positive ubiquitinated inclusions (UBIs), including frontotemporal lobar degeneration with ubiquitin-positive, tau- and α-synuclein-negative inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS).
In our lab, we have established that CaMKII-TDP overexpression at specific regions (hippocampus and cortex) transgenic mice may involve in the impairment of learning/memory capabilities by behavior test (the Morris Water Maze test). Therefore, in order to distinguish the neuronal morphology, connection and distribution at the cellular level between TDP transgene and wild type controls, we applied the technology of single-cell microinjection. In the study, we used two different dyes, Sulforhodamine101 and Biocytin, and find that there is no significant difference in total neurite length, neuritic main shaft number, neuritic branch number, and the average length of each basal neurite at the different time points. However, spine density was quantitatively measured for WT and TDP Tg of Biocytin-injected and GFP expression systems and observed that hippocampal neurons taken from TDP Tg mice grown in culture from D.I.V. 7 have fewer dendritic spines than WT controls. These findings suggest and highlight that these observations may have important implications for understanding of learning/memory decline associated with overexpression of TDP.
Abstract--------------------------------------------------1
中文摘要--------------------------------------------------2
Introduction----------------------------------------------3
Materials and Methods-------------------------------------8
Results--------------------------------------------------16
Discussion-----------------------------------------------21
References-----------------------------------------------26
Figures and Legends--------------------------------------33
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