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題名 Laminin B1抑制ERK1/2及SGK1的活化進而抑制大白鼠空間記憶的形成
Laminin B1 impairs spatial memry formation in rats through inhibition of ERK1/2 and SGK1 activity
作者 劉文婷
貢獻者 李小媛
劉文婷
關鍵詞 學習與記憶
層黏蛋白
SGK
Laminin B1
日期 2009
上傳時間 9-五月-2016 15:29:56 (UTC+8)
摘要 層黏蛋白 (laminin) 是基底膜內主要的糖蛋白，屬於細胞外基質。研究指出層黏蛋白在學習與記憶扮演重要角色，且內生性laminin β1表現於海馬迴神經，但是目前對於層黏蛋白在中樞神經的瞭解仍不是很清楚。因此，本實驗室利用水迷津學習實驗探討laminin β1對大白鼠空間學習與記憶的影響，實驗結果發現，經過學習試驗後的大白鼠，其海馬迴CA1區域的laminin β1 mRNA和蛋白質表現量明顯地減少，laminin β2的表現則不受到影響。為瞭解laminin β1在空間學習與記憶力程中扮演的角色及其下游的訊息傳遞路徑，我們轉染laminin β1 siRNA至海馬迴CA1區域會促進大白鼠在水迷津空間學習與記憶的能力，由西方墨點法也觀察到抑制海馬迴中laminin β1的表現量會促進SGK Ser422及ERK1/2的磷酸化，但是AKT Ser473的磷酸化則無顯著差異。當轉染質體基因laminin β1至CA1區域，則會抑制大白鼠水迷津空間學習與記憶的能力，同時也抑制SGK Ser422及ERK1/2的磷酸化。由於過去證實SGK1於大白鼠空間學習記憶歷程中扮演重要角色，我們推測laminin β1與SGK Ser422的磷酸化有關，為確認laminin β1與SGK1之間的關係，我們同時轉染laminin β1 siRNA和SGK siRNA至海馬迴CA1區域，原先laminin β1促進大白鼠在水迷津空間學習與記憶的能力反而受到抑制。由於許多文獻指出mitogen-activated protein kinase /extracellular signal-regulated kinase (MAPK/ERK) 能整合不同訊息傳遞路徑進而調控基因的表現，對神經突觸的可塑性及記憶的形成是個重要分子，我們進一步探討laminin β1是否透過ERK1/2調控下游的SGK1進而影響大白鼠的空間記憶與學習。因此我們先轉染laminin β1 siRNA，48小時後再給予U0126 (MEK抑制劑) 以抑制MAPK/ERK的活性。結果顯示U0126會阻斷laminin β1所引起SGK ser422的磷酸化。本篇研究提出laminin β1會參與哺乳類動物學習記憶的形成，同時也證實laminin β1會經由抑制ERK1/2及SGK1的活化進而抑制大白鼠空間學習與記憶的形成。
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描述 碩士
國立政治大學
生命科學研究所
96754005
資料來源 http://thesis.lib.nccu.edu.tw/record/#G0096754005
資料類型 thesis
dc.contributor.advisor 李小媛zh_TW
dc.contributor.author (作者) 劉文婷zh_TW
dc.creator (作者) 劉文婷zh_TW
dc.date (日期) 2009en_US
dc.date.accessioned 9-五月-2016 15:29:56 (UTC+8)-
dc.date.available 9-五月-2016 15:29:56 (UTC+8)-
dc.date.issued (上傳時間) 9-五月-2016 15:29:56 (UTC+8)-
dc.identifier (其他 識別碼) G0096754005en_US
dc.identifier.uri (URI) http://nccur.lib.nccu.edu.tw/handle/140.119/95274-
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) 96754005zh_TW
dc.description.abstract (摘要) 層黏蛋白 (laminin) 是基底膜內主要的糖蛋白，屬於細胞外基質。研究指出層黏蛋白在學習與記憶扮演重要角色，且內生性laminin β1表現於海馬迴神經，但是目前對於層黏蛋白在中樞神經的瞭解仍不是很清楚。因此，本實驗室利用水迷津學習實驗探討laminin β1對大白鼠空間學習與記憶的影響，實驗結果發現，經過學習試驗後的大白鼠，其海馬迴CA1區域的laminin β1 mRNA和蛋白質表現量明顯地減少，laminin β2的表現則不受到影響。為瞭解laminin β1在空間學習與記憶力程中扮演的角色及其下游的訊息傳遞路徑，我們轉染laminin β1 siRNA至海馬迴CA1區域會促進大白鼠在水迷津空間學習與記憶的能力，由西方墨點法也觀察到抑制海馬迴中laminin β1的表現量會促進SGK Ser422及ERK1/2的磷酸化，但是AKT Ser473的磷酸化則無顯著差異。當轉染質體基因laminin β1至CA1區域，則會抑制大白鼠水迷津空間學習與記憶的能力，同時也抑制SGK Ser422及ERK1/2的磷酸化。由於過去證實SGK1於大白鼠空間學習記憶歷程中扮演重要角色，我們推測laminin β1與SGK Ser422的磷酸化有關，為確認laminin β1與SGK1之間的關係，我們同時轉染laminin β1 siRNA和SGK siRNA至海馬迴CA1區域，原先laminin β1促進大白鼠在水迷津空間學習與記憶的能力反而受到抑制。由於許多文獻指出mitogen-activated protein kinase /extracellular signal-regulated kinase (MAPK/ERK) 能整合不同訊息傳遞路徑進而調控基因的表現，對神經突觸的可塑性及記憶的形成是個重要分子，我們進一步探討laminin β1是否透過ERK1/2調控下游的SGK1進而影響大白鼠的空間記憶與學習。因此我們先轉染laminin β1 siRNA，48小時後再給予U0126 (MEK抑制劑) 以抑制MAPK/ERK的活性。結果顯示U0126會阻斷laminin β1所引起SGK ser422的磷酸化。本篇研究提出laminin β1會參與哺乳類動物學習記憶的形成，同時也證實laminin β1會經由抑制ERK1/2及SGK1的活化進而抑制大白鼠空間學習與記憶的形成。zh_TW
dc.description.tableofcontents 致 謝 I
     中文摘要 II
     英文摘要 IV
     目 錄 VI
     圖 次 X
     縮寫全名對照表 XI
     第一章 緒 論 1
     第一節、學習與記憶 2
     一、學習與記憶的定義 2
     二、學習與記憶的種類 2
     三、記憶形成之歷程 3
     四、參與學習與記憶的腦區 4
     第二節、空間學習記憶與海馬迴組織 5
     一、海馬迴的構造與投射路徑 5
     二、海馬迴相關的動物行為模式 7
     第三節、神經細胞可塑性 8
     第四節、層黏蛋白家族 10
     一、層黏蛋白的種類 10
     二、層黏蛋白在神經系統中的表現及其功能 11
     三、層黏蛋白與記憶之間的關係 12
     第五節、血清及糖皮質激素誘導激酶 13
     一、血清及糖皮質素誘導激酶的表現及其異構型 13
     二、血清及糖皮質素誘導激酶基因表現的調控機制 15
     三、血清及糖皮質素誘導激酶訊息傳遞路徑的調控 16
     四、血清及糖皮質素誘導激酶參與學習與記憶形成的過程 18
     第六節、細胞外信號調節激酶MAPK/ERK 20
     一、細胞外信號調節激酶的家族及其異構型 20
     二、ERK1/2與學習記憶的關係 22
     第七節、本論文之研究目的與策略 24
     第二章 實驗材料與方法研究 26
     第一節 實驗動物 27
     第二節 水迷津學習試驗 27
     一、實驗儀器 27
     二、隱藏式平台 27
     三、可見式平台 29
     四、組織解剖 29
     第三節 海馬迴CA1區域內基因轉染作用 29
     一、立體定位手術 29
     二、Laminin β1質體製備 30
     三、Laminin β1 siRNA與SGK siRNA的製備 31
     四、質體基因與聚乙烯亞胺 (PEI) 混合物的製備 31
     五、藥物的製備 32
     六、海馬迴CA1區域注射 33
     第四節 西方墨點法 33
     一、蛋白質萃取 33
     二、蛋白質濃度測定法 34
     三、免疫沉澱 34
     四、西方墨點法 35
     第五節 即時定量聚合酶連鎖反應 36
     第六節 免疫組織化學染色 37
     一、組織切片的製備 37
     二、免疫組織化學染色 38
     第七節 統計分析 38
     第三章 實驗結果 40
     第一節 水迷津試驗降低海馬迴CA1區域Laminin β1 mRNA及蛋白質的表現 41
     第二節 抑制海馬迴CA1區域Laminin β1的表現會促進空間記憶的形成 46
     第三節 轉染野生型Laminin β1質體海馬迴CA1區域會抑制水迷津空間記憶的形成 51
     第四節 抑制海馬迴CA1區域Laminin β1的表現會增加ERK1/2及SGK Ser422的磷酸化 55
     第五節 轉染野生型Laminin β1質體至海馬迴CA1區域會抑制ERK1/2及SGK Ser422的磷酸化 57
     第六節 抑制海馬迴CA1區域SGK1的表現會回復Laminin β1下降所造成空間記憶促進的能力 59
     第七節 抑制海馬迴CA1區域MAPK/ERK的活化會回復Laminin β1 siRNA所促進空間學習記憶的形成，同時阻斷Laminin β1將訊息傳遞至SGK1 64
     第四章 討論 69
     第五章 結論 81
     第六章 參考資料 83
     第七章 附錄 97
     附錄一 98
     附錄二 99
     附錄三 100
     附錄四 103
     附錄五 104
     附錄六 105
     附錄七 106
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     致 謝 I
     中文摘要 II
     英文摘要 IV
     目 錄 VI
     圖 次 X
     縮寫全名對照表 XI
     第一章 緒 論 1
     第一節、學習與記憶 2
     一、學習與記憶的定義 2
     二、學習與記憶的種類 2
     三、記憶形成之歷程 3
     四、參與學習與記憶的腦區 4
     第二節、空間學習記憶與海馬迴組織 5
     一、海馬迴的構造與投射路徑 5
     二、海馬迴相關的動物行為模式 7
     第三節、神經細胞可塑性 8
     第四節、層黏蛋白家族 10
     一、層黏蛋白的種類 10
     二、層黏蛋白在神經系統中的表現及其功能 11
     三、層黏蛋白與記憶之間的關係 12
     第五節、血清及糖皮質激素誘導激酶 13
     一、血清及糖皮質素誘導激酶的表現及其異構型 13
     二、血清及糖皮質素誘導激酶基因表現的調控機制 15
     三、血清及糖皮質素誘導激酶訊息傳遞路徑的調控 16
     四、血清及糖皮質素誘導激酶參與學習與記憶形成的過程 18
     第六節、細胞外信號調節激酶MAPK/ERK 20
     一、細胞外信號調節激酶的家族及其異構型 20
     二、ERK1/2與學習記憶的關係 22
     第七節、本論文之研究目的與策略 24
     第二章 實驗材料與方法研究 26
     第一節 實驗動物 27
     第二節 水迷津學習試驗 27
     一、實驗儀器 27
     二、隱藏式平台 27
     三、可見式平台 29
     四、組織解剖 29
     第三節 海馬迴CA1區域內基因轉染作用 29
     一、立體定位手術 29
     二、Laminin β1質體製備 30
     三、Laminin β1 siRNA與SGK siRNA的製備 31
     四、質體基因與聚乙烯亞胺 (PEI) 混合物的製備 31
     五、藥物的製備 32
     六、海馬迴CA1區域注射 33
     第四節 西方墨點法 33
     一、蛋白質萃取 33
     二、蛋白質濃度測定法 34
     三、免疫沉澱 34
     四、西方墨點法 35
     第五節 即時定量聚合酶連鎖反應 36
     第六節 免疫組織化學染色 37
     一、組織切片的製備 37
     二、免疫組織化學染色 38
     第七節 統計分析 38
     第三章 實驗結果 40
     第一節 水迷津試驗降低海馬迴CA1區域Laminin β1 mRNA及蛋白質的表現 41
     第二節 抑制海馬迴CA1區域Laminin β1的表現會促進空間記憶的形成 46
     第三節 轉染野生型Laminin β1質體海馬迴CA1區域會抑制水迷津空間記憶的形成 51
     第四節 抑制海馬迴CA1區域Laminin β1的表現會增加ERK1/2及SGK Ser422的磷酸化 55
     第五節 轉染野生型Laminin β1質體至海馬迴CA1區域會抑制ERK1/2及SGK Ser422的磷酸化 57
     第六節 抑制海馬迴CA1區域SGK1的表現會回復Laminin β1下降所造成空間記憶促進的能力 59
     第七節 抑制海馬迴CA1區域MAPK/ERK的活化會回復Laminin β1 siRNA所促進空間學習記憶的形成，同時阻斷Laminin β1將訊息傳遞至SGK1 64
     第四章 討論 69
     第五章 結論 81
     第六章 參考資料 83
     第七章 附錄 97
     附錄一 98
     附錄二 99
     附錄三 100
     附錄四 103
     附錄五 104
     附錄六 105
     附錄七 106		zh_TW
dc.source.uri (資料來源) http://thesis.lib.nccu.edu.tw/record/#G0096754005en_US
dc.subject (關鍵詞) 學習與記憶zh_TW
dc.subject (關鍵詞) 層黏蛋白zh_TW
dc.subject (關鍵詞) SGKen_US
dc.subject (關鍵詞) Laminin B1en_US
dc.title (題名) Laminin B1抑制ERK1/2及SGK1的活化進而抑制大白鼠空間記憶的形成zh_TW
dc.title (題名) Laminin B1 impairs spatial memry formation in rats through inhibition of ERK1/2 and SGK1 activityen_US
dc.type (資料類型) thesisen_US
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