Title page for 952211012


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Student Number 952211012
Author Jheng-Jie Kang(康証傑)
Author's Email Address No Public.
Statistics This thesis had been viewed 1707 times. Download 1907 times.
Department Graduate Institute of Systems Biology and Bioinformatics
Year 2007
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Using PC-12 cell line to establish a molecular and cellular biological model for studying neuronal signaling transduction
Date of Defense 2008-07-17
Page Count 61
Keyword
  • PC-12
  • Pheochromocytoma
  • transcription factor
  • Abstract Cloned rat pheochromocytoma (PC-12) cell has been used as in vitro model of neuroscience research for a long time since it could differentiate both morphologically and
    biologically into neuronal cells. However, the ratio of undifferentiated to differentiated PC-12 cells can have noises that affect the outcomes of the experiments. In this study, we attempt to enhance the degree of PC-12 cell differentiation, repress the proliferative ability of
    undifferentiated PC-12 cells and promote the ratio of differentiated PC-12 to whole cells by changing the cell culture condition. In addition, we apply this model to screen the variation of the nuclear transcription factors (TFs). We choose the Glutamate induced NMDA receptor
    model in our study of neuronal activity signaling pathway and systematically studied 345 TFs by protein/DNA array. Our result indicates that except for the several known TFs, which have been shown to increase the affinity or content, there were more TFs, which were uncovered
    and also have good affinity. In this experiment, we established a neuron-like cell model, which has a relative higher degree of differentiation and confirmed the specificity of NMDA receptor to Glutamate.
    Table of Content 中文提要 …………………………………………………………… i
    英文提要 …………………………………………………………… ii
    誌謝 …………………………………………………………… iii
    目錄 …………………………………………………………… iv
    一、  緒論…………………………………………………………1
    1-1  前言…………………………………………………………1
    1-2  嗜鉻性細胞瘤細胞(PC-12)及其分化…………………… 1
    1-2-1 嗜鉻性細胞瘤細胞株………………………………………1
    1-2-2 神經生長因子………………………………………………2
    1-3  神經細胞模型種類與培養基………………………………3
    1-3-1 神經細胞模型種類…………………………………………3
    1-3-2 培養液種類…………………………………………………4
    1-3-3 神經細胞基礎培養液………………………………………4
    1-4  麩胺酸誘導之NMDA 受體活化…………………………… 5
    1-4-1 興奮性神經傳導物質麩胺酸及其受體……………………5
    1-4-2 NMDA 受體及其功能……………………………………… 6
    1-5  神經活化即轉錄因子調控…………………………………8
    1-6  研究緣起……………………………………………………9
    二、  實驗材料與方法……………………………………………11
    2-1  細胞培養預藥物處理………………………………………11
    2-1-1 細胞培養載具材料之處理…………………………………11
    2-1-2 細胞培養……………………………………………………11
    2-1-3 藥物處理……………………………………………………11
    2-1-3-1PC-12 細胞分盤播種………………………………………12
    2-1-3-2PC-12 細胞神經生長因子處理……………………………12
    2-1-4 麩胺酸處理…………………………………………………13
    2-1-4-1PC-12 之麩胺酸處理………………………………………13
    2-1-4-2類神經細胞之麩胺酸處理…………………………………13
    2-2  基因表現分析………………………………………………13
    2-2-1 細胞RNA 萃取………………………………………………13
    2-2-2 反轉錄聚合酶連鎖反應…………………………………… 14
    2-2-3 螢光即時定量聚合酶連鎖反應…………………………… 15
    2-2-3-1反轉錄反應………………………………………………… 15
    2-2-3-2聚合酶連鎖反應…………………………………………… 15
    2-3  細胞免疫螢光染色………………………………………… 16
    2-4  蛋白質分析………………………………………………… 16
    2-4-1 蛋白質萃取………………………………………………… 16
    2-4-1-1細胞質蛋白萃取…………………………………………… 16
    2-4-1-2細胞核蛋白萃取…………………………………………… 17
    2-4-2 BCA 定量分析.………………………………………………17
    2-4-3 SDS-PAGE 膠體置備…………………………………………18
    2-4-4 蛋白質樣品前處理………………………………………… 18
    2-4-5 蛋白質電永操作…………………………………………… 18
    2-4-6 半濕式蛋白質轉印法……………………………………… 19
    2-4-7 免疫染色…………………………………………………… 19
    2-4-8 轉錄因子核酸矩陣列分析………………………………… 20
    2-5  研究使用軟體……………………………………………… 21
    三、  結果………………………………………………………… 22
    四、  討論 ……………………………………………………… 41
    4-1 大鼠嗜鉻性瘤細胞之分化…………………………………… 41
    4-2 類神經PC-12 細胞於NMDA 受體功能性之探討 …………… 43
    4-3 轉錄因子-核酸陣列分析……………………………………  44
    五、 結論…………………………………………………………… 46
    參考文獻………………………………………………………………47
    附錄一DMEM 培養液與Neurobasal 培養液內容物差別列表………51
    附錄二 反轉錄酶聚合酶連鎖反應所用引子列表………………… 52
    附錄三 即時定量聚合酶連鎖反應所用引子探針列表…………… 52
    附錄四 轉錄因子陣列實驗流程圖………………………………… 53
    附錄五 轉錄因子陣列……………………………………………… 54
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  • Qing-Dong Ling(凌慶東)
  • Hoong-Chien Lee(李弘謙)
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    Date of Submission 2008-07-22

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