博碩士論文 952211012 詳細資訊


姓名 康証傑(Jheng-Jie Kang)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 以大鼠嗜鉻性瘤細胞株建立神經訊號傳遞之細胞分子生物學模型
(Using PC-12 cell line to establish a molecular and cellular biological model for studying neuronal signaling transduction)
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摘要(中) PC-12細胞由於具有被神經生長因子﹝NGF﹞誘導分化為類神經細胞的特性,長期以來作為神經科學相關研究的體外模型被廣泛運用。然而在最終所運用於實驗的類神經PC-12細胞中由於分化率的限制,未分化PC-12 細胞對實驗結果所可能造成之難以避免雜訊,一直以來是一個困擾的問題。本實驗利用細胞培養環境的改變,以提高已分化類神經 PC-12細胞的分化程度、降低未分化 PC-12 細胞的增殖能力、提升類神經 PC-12 細胞在整體細胞中的比例,並用此模型作為實驗平台,通過觀察核內基因轉錄因子的變化,進行神經活化後訊息傳遞的研究。在神經活化的模型上,我們選擇麩胺酸(Glutamic acid)活化 NMDA受體引發胞內分子訊息傳遞的模型,對已知 345 個轉錄因子進行轉錄因子-核酸矩陣列實驗之系統性研究。在麩胺酸刺激後之類神經細胞轉錄因子的觀察中,我們發現除了過去較為已知數個轉錄因子具有親和力(affinity)或含量的提升,尚有更多的轉錄因子也具有兩倍以上的提升量。另外在神經生長因子誘導下,意外發現多數轉錄因子在誘導分化之後呈現親和力或含量的下降。在我們的研究結果中,我們不但建立了一個類神經 PC-12 細胞分化程度相對較高的神經細胞模型,並且也確認了其NMDA受體對麩胺酸刺激的專一性能力。本研究結果為對神經細胞內分子訊息傳遞研究建構了一個可實際應用的實驗平台,為將來更進一步的細胞內分子生物模型研究打下良好的基礎。
摘要(英) 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.
關鍵字(中) ★ 嗜鉻性 轉錄因子 關鍵字(英) ★ PC-12
★ transcription factor
★ Pheochromocytoma
論文目次 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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指導教授 凌慶東、李弘謙
(Qing-Dong Ling、Hoong-Chien Lee)
審核日期 2008-7-22

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