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研究生： 吳若羚
Wu, Jo-Ling
論文名稱： 結合Lab-on-a-chip與二維蛋白質電泳技術進行海馬迴神經細胞及神經軸突蛋白質組成分析
Using lab-on-a-chip platform and two-dimensional gel electrophoresis fpr analyzing neuronal whole cell and axon proteome
指導教授： 張兗君
Chang, Yen-Chung
口試委員: 周韻家
詹鴻霖
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2011
畢業學年度： 99
語文別： 中文
論文頁數： 67
中文關鍵詞： 海馬迴神經細胞 、蛋白質體學
相關次數： 點閱：2  下載：0 
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    • 過去的研究認為，神經細胞所需要的蛋白質是由細胞本體轉譯合成後再送至目的地，但是最近有許多研究指出，雖然神經軸突內主要的蛋白質是由細胞體合成，但仍有部份蛋白質是透過局部蛋白質合成作用產生。因此，為了比較神經細胞與神經軸突蛋白質組成的不同，本實驗發展一種神經細胞培養裝置 (neuronal chip device)，其可以侷限體生長，並引導神經軸突向同一方向前進至另一區塊。此裝置軸突生長區域的下方設計有一道細微的紋溝，藉由這道紋溝可以將神經軸突與其細胞體及樹突進行有效率的分離，故此神經細胞培養裝置可以大量的收集神經軸突蛋白質，以進行二維蛋白質電泳分析。
    本篇研究首先進行二維蛋白質電泳分析條件之改進，並建立出適合分析神經細胞及神經軸突蛋白質的條件；我們使用這些改善後的條件對神經細胞及神經軸突的蛋白質組成進行分析，並透過二維蛋白質分析技術，發現神經細胞及神經軸突皆含有特異性的蛋白質，未來可以搭配 MALDI-TOF 針對這些蛋白質進行鑑定。

    中文摘要 ................................................................................................................... i 英文摘要 .................................................................................................................. ii 謝誌 ......................................................................................................................... iii 目錄 ......................................................................................................................... iv 壹、緒論 .................................................................................................................. 1 貳、材料與方法 ....................................................................................................... 6 一、印章 (Stamp)的設計與製作 ......................................................................... 6 1、圖形設計 ..................................................................................................... 6 2、模仁 (Mold)的製作 ..................................................................................... 6 3、印章 (Stamp)的製作 ................................................................................... 7 4、PDMS 模板 (Stencil)的製作 ...................................................................... 8 5、刻痕玻片 ..................................................................................................... 8 6、對準系統(Alignment system) ....................................................................... 9 二、微接觸壓印 (Microcontact printing) ............................................................. 9 三、初代海馬迴神經細胞培養 ............................................................................ 9 1、解剖大鼠取胚鼠海馬迴神經細胞 ............................................................... 9 2、海馬迴神經細胞培養 ................................................................................ 10 四、蛋白質萃取 ................................................................................................. 11 1、海馬迴神經細胞 (Neurol whole cell)去細胞核的蛋白質萃取 ................. 11 2、海馬迴神經細胞 (Neurol whole cell) 蛋白質萃取 ................................... 12 3、神經軸突 (Axon)蛋白質萃取 ................................................................... 12 五、蛋白質濃度測定.......................................................................................... 13 1、海馬迴神經細胞 (Neurol whole cell) 蛋白質 .......................................... 13 2、神經軸突 (Axon)蛋白質 ........................................................................... 14 六、凝膠電泳分析 (SDS-PAGE)....................................................................... 14 七、丙酮沉澱 (Acetone precipitation) ............................................................... 15 八、二維蛋白質電泳 (Two-dimensional gel electrophoresis) ............................ 15 1、第一維電泳 ................................................................................................ 15 2、第二維電泳 ................................................................................................ 16 九、銀染色法(Sliver staining) ............................................................................ 17 1、Hong-Lin Chan等人使用之銀染法 ........................................................... 17 2、Farzin Gharahdaghi等人發明之染色法 .................................................... 17 3、Amersham Biosciences公司提供 .............................................................. 18 十、Coomassie Brilliant Blue 染色法 ................................................................ 19 1、本實驗室沿用之 Coomassie Brilliant Blue 染色法 .................................. 19 2、Volker Neuhoff 等人發明之 Coomassie Brilliant Blue 染色法 ............... 19 十一、西方墨點法 (Western blot) ................................................................... 19 十二、免疫螢光染色 (Immunocytochemistry) .................................................. 20 參、結果 ................................................................................................................ 22 一、海馬迴神經軸突蛋白質收集之純化鑑定 ................................................... 22 二、二維蛋白質電泳最佳條件的選擇及改善 ................................................... 23 1、第一維電泳 (IPGphor isoelectric focusing system) 條件選擇 .................. 23 2、樣品處理之改善 ........................................................................................ 24 3、染色方法之改善 ........................................................................................ 27 三、海馬迴神經細胞蛋白質集軸突蛋白質分析比較........................................ 29 1、利用一維膠凝電泳分析海馬迴神經細胞及海馬迴神經次細胞 (軸 突)蛋白質之組成 ..................................................................................................... 29 2、利用二維蛋白質電泳分析海馬迴神經細胞及海馬迴神經次細胞 (軸突)蛋白質之組成 ..................................................................................................... 30 肆、討論 ................................................................................................................ 33 一、神經細胞與次細胞分離鑑定 ...................................................................... 33 二、二維蛋白質電泳分析探討 .......................................................................... 34 三、神經細胞次單位結構之蛋白質分析比較 ................................................... 36 伍、文獻參考 ......................................................................................................... 37 六、圖表 ................................................................................................................ 41 圖一、海馬迴神經軸突蛋白質收集之純化鑑定 ............................................... 41 圖二、第一維電泳條件選擇 .............................................................................. 42 圖三、去細胞核對二維蛋白質電泳的影響 ....................................................... 44 圖四、Tris 對二維蛋白質電泳的影響 .............................................................. 45 圖五、丙酮沉澱對二維蛋白質電泳的影響 ....................................................... 46 圖六、二維蛋白質電泳之靈敏度測詴 ............................................................... 47 圖七、銀離子染色法之改善 .............................................................................. 48 圖八、Coomassie Brilliant Blue染色法之改善 .................................................. 49 圖九、利用一維膠凝電泳分析海馬迴神經細胞及海馬迴神經次細胞 (軸突)蛋白質之組成 ..................................................................................................... 51 圖十、以二維蛋白質電泳分析神經細胞的蛋白質樣品 .................................... 52 圖十一、以二維蛋白質電泳分析神經軸突的蛋白質樣品 ................................ 53 圖十二、利用二維膠凝電泳分析海馬迴神經細胞及海馬迴神經次細胞(軸突)蛋白質之組成 ......................................................................................................... 55 圖十三、利用3D顯示神經細胞與神經軸突中具差異性的蛋白質點 .............. 57 表一、神經細胞蛋白質點分析 .......................................................................... 58 表二、神經軸突蛋白質點分析 .......................................................................... 61 表三、神經細胞與神經軸突間重複性蛋白質點分析........................................ 64 附錄一、神經細胞培養基板裝置 (neuronal chip device) ................................ 65 附錄二、ImageMaster 2D Platinum 7.0軟體數值分析標準 .............................. 67

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