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題名 確認PIAS1在促進大鼠空間學習與記憶的嶄新角色之探討
Identification of a novel role of PIAS1 in facilitation of spatial memory formation in rats
作者 劉彥呈
貢獻者 李小媛
劉彥呈
關鍵詞 空間學習與記憶
莫氏水迷津試驗
西方墨點法
海馬迴
Spatial learning and memory
Morris water maze
Western blot
Hippocampus
PIAS1
STAT1
日期 2009
上傳時間 9-May-2016 15:29:54 (UTC+8)
摘要 本實驗室於先前利用莫氏水迷津試驗篩選學習快與學習慢的大白鼠，取出其海馬迴組織並進行聚合酶連鎖反應差異顯示(PCR differential display)，結果顯示學習快與學習慢的大白鼠背側海馬迴之間共有98個cDNA片段有差異表現。把這些cDNA片段進行定序並利用BLAST資料庫比對，其中一個cDNA片段為大白鼠的pias1 [protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1)] 基因。為了瞭解pias1基因的表現是否和空間學習有所關聯，隨機把大白鼠分成兩組，一組為有訓練組別(有空間線索與隱藏式平台)，另一組為無訓練的組別(沒有平台，作為游泳的控制組)同時進行莫氏水迷津學習試驗。試驗完畢，取出海馬迴組織進行即時定量聚合酶連鎖反應與西方墨點法來分析PIAS1的mRNA與蛋白質的表現。結果顯示有水迷津訓練的大白鼠，其PIAS1的mRNA與蛋白質表現皆明顯的高於無訓練的組別。為了更進一步確認PIAS1在空間學習中所扮演的角色，我們利用基因轉染的技術，轉染PIAS1 siRNA至大白鼠海馬迴CA1區域抑制PIAS1的表現。我們發現轉染PIAS1 siRNA至CA1區域會抑制大白鼠在水迷津的行為表現，然而轉染野生型的PIAS1質體基因至CA1區域卻會增進水迷津試驗的學習能力，同時我們也以西方墨點法發現，當轉染PIAS1 siRNA會增加STAT1 Tyr701的磷酸化，而轉染PIAS1 WT則會抑制STAT1 Tyr701的磷酸化。為了探討PIAS1促進記憶形成的分子機制，我們發現當轉染突變型的STAT1 Y701F質體基因至CA1區域，會抑制PIAS1 siRNA所造成記憶的損害。這些實驗結果代表著PIAS1會抑制STAT1 Tyr701的磷酸化，而PIAS1促進記憶的形成可能是藉由抑制STAT1 Tyr701的磷酸化而達成。另外，我們也單獨轉染突變型的STAT1 Y701F質體基因至CA1區域，水迷津實驗結果顯示會促進空間記憶的形成。目前PIAS1在免疫的角色已有許多研究證實，但是本篇研究是第一個提出PIAS1會參與哺乳類動物學習與記憶形成探討。
Our laboratory has previously identified 98 cDNA fragments by using PCR differential display from rat dorsal hippocampus that are differentially expressed between fast learners and slow learners from the water maze learning task. After sequencing and BLAST analysis, one of these cDNA fragments encodes the rat pias1 [protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1)] gene. In order to determine whether pias1 gene expression is associated with spatial learning, naïve rats were randomly assigned to the trained group (with visual cues and platform been present) and the non-trained group (without the platform as the swimming control). The dorsal hippocampus from these animals was dissected out at the end of the training and was subjected to RNA and protein extraction for real-time PCR and Western blot analysis of PIAS1 expression, respectively. Results revealed that the pias1 mRNA level and protein level was both higher in the hippocampus of trained rats than non-trained rats. To further examine the role of PIAS1 involved in spatial learning and memory, the specific PIAS1 siRNA was used to knockdown the expression of PIAS1 in rat hippocampal CA1 region. We found that transfection of PIAS1 siRNA to the CA1 area impaired water maze performance, whereas transfection of the wild-type PIAS1 DNA plasmid to the CA1 area facilitated water maze performance in rats. Meanwhile, PIAS1 siRNA increased STAT1 phosphorylation at Tyr701 whereas PIAS1 WT decreased STAT1 phosphorylation at this residue. In the examination of molecular mechanism underlying PIAS1-mediated memory facilitation, we have found that transfection of the STAT1 Y701F mutant plasmid antagonized the memory-impairing effect of PIAS1 siRNA, whereas transfection of STAT1 Y701F alone facilitated spatial memory formation. These results together suggest that one of the molecular mechanisms underlying PIAS1-mediated memory facilitation is through decreased STAT1 phosphorylation at Tyr701. All these manipulations did not affect visible platform learning in rats. In addition to the well documented role of PIAS1 in the immune system, here we have been the first to demonstrate a novel role of PIAS1 involved in spatial memory formation in rats.
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描述 碩士
國立政治大學
生命科學研究所
96754001
資料來源 http://thesis.lib.nccu.edu.tw/record/#G0096754001
資料類型 thesis
dc.contributor.advisor 李小媛zh_TW
dc.contributor.author (Authors) 劉彥呈zh_TW
dc.creator (作者) 劉彥呈zh_TW
dc.date (日期) 2009en_US
dc.date.accessioned 9-May-2016 15:29:54 (UTC+8)-
dc.date.available 9-May-2016 15:29:54 (UTC+8)-
dc.date.issued (上傳時間) 9-May-2016 15:29:54 (UTC+8)-
dc.identifier (Other Identifiers) G0096754001en_US
dc.identifier.uri (URI) http://nccur.lib.nccu.edu.tw/handle/140.119/95273-
dc.description (描述) 碩士zh_TW
dc.description (描述) 國立政治大學zh_TW
dc.description (描述) 生命科學研究所zh_TW
dc.description (描述) 96754001zh_TW
dc.description.abstract (摘要) 本實驗室於先前利用莫氏水迷津試驗篩選學習快與學習慢的大白鼠，取出其海馬迴組織並進行聚合酶連鎖反應差異顯示(PCR differential display)，結果顯示學習快與學習慢的大白鼠背側海馬迴之間共有98個cDNA片段有差異表現。把這些cDNA片段進行定序並利用BLAST資料庫比對，其中一個cDNA片段為大白鼠的pias1 [protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1)] 基因。為了瞭解pias1基因的表現是否和空間學習有所關聯，隨機把大白鼠分成兩組，一組為有訓練組別(有空間線索與隱藏式平台)，另一組為無訓練的組別(沒有平台，作為游泳的控制組)同時進行莫氏水迷津學習試驗。試驗完畢，取出海馬迴組織進行即時定量聚合酶連鎖反應與西方墨點法來分析PIAS1的mRNA與蛋白質的表現。結果顯示有水迷津訓練的大白鼠，其PIAS1的mRNA與蛋白質表現皆明顯的高於無訓練的組別。為了更進一步確認PIAS1在空間學習中所扮演的角色，我們利用基因轉染的技術，轉染PIAS1 siRNA至大白鼠海馬迴CA1區域抑制PIAS1的表現。我們發現轉染PIAS1 siRNA至CA1區域會抑制大白鼠在水迷津的行為表現，然而轉染野生型的PIAS1質體基因至CA1區域卻會增進水迷津試驗的學習能力，同時我們也以西方墨點法發現，當轉染PIAS1 siRNA會增加STAT1 Tyr701的磷酸化，而轉染PIAS1 WT則會抑制STAT1 Tyr701的磷酸化。為了探討PIAS1促進記憶形成的分子機制，我們發現當轉染突變型的STAT1 Y701F質體基因至CA1區域，會抑制PIAS1 siRNA所造成記憶的損害。這些實驗結果代表著PIAS1會抑制STAT1 Tyr701的磷酸化，而PIAS1促進記憶的形成可能是藉由抑制STAT1 Tyr701的磷酸化而達成。另外，我們也單獨轉染突變型的STAT1 Y701F質體基因至CA1區域，水迷津實驗結果顯示會促進空間記憶的形成。目前PIAS1在免疫的角色已有許多研究證實，但是本篇研究是第一個提出PIAS1會參與哺乳類動物學習與記憶形成探討。zh_TW
dc.description.abstract (摘要) Our laboratory has previously identified 98 cDNA fragments by using PCR differential display from rat dorsal hippocampus that are differentially expressed between fast learners and slow learners from the water maze learning task. After sequencing and BLAST analysis, one of these cDNA fragments encodes the rat pias1 [protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1)] gene. In order to determine whether pias1 gene expression is associated with spatial learning, naïve rats were randomly assigned to the trained group (with visual cues and platform been present) and the non-trained group (without the platform as the swimming control). The dorsal hippocampus from these animals was dissected out at the end of the training and was subjected to RNA and protein extraction for real-time PCR and Western blot analysis of PIAS1 expression, respectively. Results revealed that the pias1 mRNA level and protein level was both higher in the hippocampus of trained rats than non-trained rats. To further examine the role of PIAS1 involved in spatial learning and memory, the specific PIAS1 siRNA was used to knockdown the expression of PIAS1 in rat hippocampal CA1 region. We found that transfection of PIAS1 siRNA to the CA1 area impaired water maze performance, whereas transfection of the wild-type PIAS1 DNA plasmid to the CA1 area facilitated water maze performance in rats. Meanwhile, PIAS1 siRNA increased STAT1 phosphorylation at Tyr701 whereas PIAS1 WT decreased STAT1 phosphorylation at this residue. In the examination of molecular mechanism underlying PIAS1-mediated memory facilitation, we have found that transfection of the STAT1 Y701F mutant plasmid antagonized the memory-impairing effect of PIAS1 siRNA, whereas transfection of STAT1 Y701F alone facilitated spatial memory formation. These results together suggest that one of the molecular mechanisms underlying PIAS1-mediated memory facilitation is through decreased STAT1 phosphorylation at Tyr701. All these manipulations did not affect visible platform learning in rats. In addition to the well documented role of PIAS1 in the immune system, here we have been the first to demonstrate a novel role of PIAS1 involved in spatial memory formation in rats.en_US
dc.description.tableofcontents 目 錄..................................................I
     圖 次..................................................IV
     縮寫對照表...............................................V
     中文摘要................................................VII
     英文摘要................................................IX
     第一章 緒 論.............................................1
     第一節、學習與記憶 ........................................1
      一、學習與記憶的定義...................................1
      二、學習與記憶的種類.....................................1
      三、參與學習與記憶的腦區..................................2
     第二節、海馬迴.............................................3
      一、海馬迴的結構與神經投射路徑.............................3
      二、海馬迴與學習記憶的關係.................................5
     第三節、學習記憶與長期增益效應的相關性........................6
      一、長期增益效應形成的機制.................................7
      二、長期增益效應與記憶的相關性..............................8
     第四節、PIAS蛋白質家族簡介...................................9
     第五節、PIAS家族蛋白所媒介的基因調控機制......................12
      一、PIAS抑制轉錄因子活性的調控機制.........................12
      二、PIAS促進轉錄因子活性的調控機制.........................15
     第六節、PIAS調節STATs的訊息傳遞.............................17
     第七節、訊息傳遞與轉錄活化因子家族簡介........................17
     第八節、訊息傳遞與轉錄活化因子在細胞生理功能所扮演的角色........21
     第九節、本論文之研究目的與策略...............................22
     第二章 材料與方法..........................................23
     第一節、實驗動物...........................................23
     第二節、海馬迴內基因轉染作用................................23
      一、立體定位手術與埋管.................................23
      二、野生型pCMV-tag2A-PIAS1質體基因的建構...............24
      三、突變型pRC/CMV-flag-STAT1 alpha質體基因的建構.......25
      四、質體基因與聚乙烯亞胺混合物的製備.....................26
      五、PIAS1小干擾RNA(PIAS1 siRNA)的製備.................26
      六、海馬迴CA1區域注射..................................27
     第三節、免疫組織化學染色....................................27
      一、組織切片的製備.......................................27
      二、免疫組織化學染色.....................................28
     第四節、動物行為實驗：莫氏水迷津學習試驗......................29
      一、水迷津實驗器材.......................................29
      二、隱藏式平台水迷津學習試驗...............................29
      三、可見式平台水迷津學習試驗...............................32
     第五節、西方墨點法..........................................32
      一、分離海馬迴、紋狀體及杏仁核組織..........................33
      二、腦組織蛋白質萃取......................................33
      三、蛋白質濃度測定法......................................34
      四、西方墨點法...........................................34
     第六節、即時定量聚合酶連鎖反應...............................36
     第七節、細胞實驗............................................37
      一、細胞培養.............................................37
      二、細胞轉染小干擾RNA.....................................38
     第八節、統計分析............................................39
     第三章 實驗結果............................................40
     第一節、聚合酶連鎖反應差異顯示法篩選與空間學習記憶有關的基因.....40
     第二節、水迷津試驗能促使海馬迴的PIAS1表現量增加................42
     第三節、轉染野生型PIAS1至海馬迴區域會促進水迷津空間記憶的形成....46
     第四節、海馬迴轉染野生型PIAS1會抑制內生型STAT1的磷酸化.........50
     第五節、PIAS1是空間學習記憶形成的重要蛋白質...................52
     第六節、抑制PIAS1的表現會增加STAT1 Tyr701的磷酸化............56
     第七節、抑制STAT1 Tyr701的磷酸化會促進水迷津空間記憶的形成.....58
     第八節、轉染STAT1 Y701F至海馬迴會促進水迷津空間記憶的形成......62
     第九節、細胞轉染PIAS1 siRNA確認STAT1 Tyr701磷酸化的作用機制...66
     第四章 討 論..............................................68
     第五章 結 論..............................................77
     參考文獻..................................................78
     附 錄....................................................84
     附錄一、..................................................84
     附錄二、..................................................85
     附錄三、..................................................86
     附錄四、..................................................87
     附錄五、..................................................88		zh_TW
dc.source.uri (資料來源) http://thesis.lib.nccu.edu.tw/record/#G0096754001en_US
dc.subject (關鍵詞) 空間學習與記憶zh_TW
dc.subject (關鍵詞) 莫氏水迷津試驗zh_TW
dc.subject (關鍵詞) 西方墨點法zh_TW
dc.subject (關鍵詞) 海馬迴zh_TW
dc.subject (關鍵詞) Spatial learning and memoryen_US
dc.subject (關鍵詞) Morris water mazeen_US
dc.subject (關鍵詞) Western bloten_US
dc.subject (關鍵詞) Hippocampusen_US
dc.subject (關鍵詞) PIAS1en_US
dc.subject (關鍵詞) STAT1en_US
dc.title (題名) 確認PIAS1在促進大鼠空間學習與記憶的嶄新角色之探討zh_TW
dc.title (題名) Identification of a novel role of PIAS1 in facilitation of spatial memory formation in ratsen_US
dc.type (資料類型) thesisen_US
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