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系統識別號 U0026-0812200914363625
論文名稱(中文) 於去氧去醣中風模式下的神經性細胞存活率
論文名稱(英文) The Viability of Neuronal Cells through an Oxygen-glucose Deprivation Stroke Model
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 96
學期 2
出版年 97
研究生(中文) 蔡守來
研究生(英文) Shou-Lai Tsai
學號 P8694125
學位類別 碩士
語文別 英文
論文頁數 41頁
口試委員 指導教授-陳家進
口試委員-葉明龍
口試委員-張憲彰
中文關鍵字 PC12細胞株  去氧去醣  微培養箱  缺血性中風模型 
英文關鍵字 micro incubator  oxygen-glucose deprivation (OGD)  ischemic stroke model  PC12 
學科別分類
中文摘要 長期以來中風在工業化國家是主要的死亡與失能原因之一。試管內缺血性中風模型比活體模型更能精確地檢測影響病理生理學進程的因素。在試管內缺血性中風模型中去氧去醣的方法是普遍被使用的。加入神經生長因子後外觀會和神經元相似的PC12細胞株已經被用來從事許多與神經細胞相關的研究。為了連續觀察細胞,有些特別的培養箱已經被發展出來。微培養箱是這些特別的培養箱之一,它不只能直接連續觀察細胞,並因為裝置的體積較小而耗能較低、試劑用量較少而花費較少,而且能得到快速的反應。本研究架設了一個由本實驗室自製的微電極改造的微培養系統以從事去氧去醣中風實驗,並且以螢光染色研究PC12細胞株在這個去氧去醣中風環境下的存活率以驗證此系統。細胞數量經由數位影像處理來計算。細胞的平均存活率是去氧去醣前99.29±0.14%,去氧去醣後30分鐘後53.50±5.83%,回氧回醣24小時後0.00±0.00%。螢光測試的影響因素,包括雙色螢光劑的適當比例與濃度、將螢光劑染上細胞的適當培養時間與溫度、避免螢光雜質的方法、適合計算細胞數的細胞分佈、減緩螢光模糊掉的方法,都詳細地討論與解決了。此去氧去醣中風系統將可與微電極陣列的電生理學量測合併,包括螢光測試與電生理學量測,都可進行長時間的間隔定時記錄。
英文摘要 Stroke has long been a major cause of death and disability in industrialized nations. In-vitro ischemic stroke models allow for a more rigorous examination of the factors that influence pathophysiological process achieved in-vivo study. Oxygen-glucose deprivation (OGD) method is commonly used for in-vitro ischemic stroke models. PC12 cell lines, which would appear like neurons by treated with nerve growth factor, have been used to approach a number of fundamental problems related to neuron cells. For the continuous observation of cells, some specific incubators have been developed. Micro incubator, one of these specific incubators, could not only enable observation of cells directly, but also cost less for its low consumption of power and reagents. It also provides fast response due to the small device volume. In this study, a micro-incubation system for OGD experiments incorporated with our previous self-made microelectrode array (MEA) was established. The PC12 cell viability through this OGD system was investigated with fluorescent test before OGD, after OGD 30 minutes, and after recovery 24 hours to confirm our model. Image processing techniques were utilized to count cell numbers. The average living cell percentages were 99.29±0.14% before OGD, 53.50±5.83% after OGD, but almost completely damage during recovery phase. Considerations for fluorescent test were discussed and solved, including appropriate concentration and proportion of the two-color fluorescent kit, suitable incubating time and temperature, impurities avoidance, proper cell distribution for counting, and minimizing blur of fluorescence. In the future, this OGD system could be merged with electro-plysiological recording of micro-electrode array (MEA). Both fluorescent test and electro-plysiological recording could be achieved time-lapse recording.
論文目次 中文摘要………………………………………………………………I
Abstract………………………………………………………………II
誌謝……………………………………………………………………III
Content………………………………………………………………IV
List of Tables………………………………………………………VI
List of Figures……………………………………………………VII
Chapter 1 Introduction……………………………………………1
1.1 Introduction to ischemic stroke……………………1
1.2 In-vitro models for ischemic stroke: oxygen-glucose deprivation (OGD) …2
1.2.1 Purpose of in-vitro model……………………………2
1.2.2 General principles of culturing nerve cells……2
1.2.3 Induction of in-vitro ischemia though OGD………3
1.2.4 PC12 cell line…………………………………………5
1.3 Micro-incubation for tissue culture………………5
1.3.1 Purpose of micro-incubation…………………………5
1.3.2 Considerations of micro-incubation………………6
1.4 Introduction to micro-electrode array……………9
1.5 The aim of this study…………………………………10
Chapter 2 Materials and Methods………………………………11
2.1 Cell culture……………………………………………11
2.2 Fabrication of Petri-dishlike microelectorde array…11
2.3 Experimental setup……………………………………12
2.4 Oxygen-glucose deprivation (OGD)…………………16
2.4.1 Seeding cells on MEA…………………………………16
2.4.2 OGD medium preparation………………………………17
2.4.3 Induction of OGD………………………………………19
2.5 Fluorescent test………………………………………20
Chapter 3 Results…………………………………………………22
3.1 De-oxygenation of OGD medium………………………22
3.2 Determination of incubating time for fluorescent test…22
3.3 Fluorescent images through OGD……………………23
3.4 Image processing for cell counting………………27
3.5 Viability of PC12 cells through OGD……………29
3.6 Control group…………………………………………30
Chapter 4 Discussion and Conclusion…………………………33
4.1 Confirmation of our OGD system…………………………33
4.2 Considerations for fluorescent test……………………34
4.3 Conclusion and future development………………………35
References……………………………………………………………37
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