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研究生:王若亭
研究生(外文):Wang, Jo-Ting
論文名稱:即時螢光細胞生長監控儀之開發與生醫研究應用
論文名稱(外文):Developing and applying a real-time fluorescence cell growth monitoring system on biomedical study
指導教授:張格東
指導教授(外文):Chang, Ko-Tung
口試委員:邱紹智王耀男
口試委員(外文):Chiu, Shao-ChihWang, Yao-Nan
口試日期:2016-01-25
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技系所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:63
中文關鍵詞:CFSE細胞增生轉染RAW 264.7即時螢光
外文關鍵詞:CFSEcell proliferationtransfectionRAW 264.7real-timefluorescence
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  • 下載下載:4
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CMOS (Complementary Metal-Oxide-Semiconductor) 即時螢光細胞生長監控儀可應用於生物活性試驗,像是偵測細胞轉染效率、細胞增生、活性氧物質或是新開發藥物之細胞毒性試驗。本研究目的將藉由這些生物試驗證實我們所開發CMOS即時螢光細胞生長監控儀系統之可行性與準確性。在細胞增生實驗上,使用CFSE螢光染劑標定RAW 264.7細胞進行培養,藉由新開發之儀器分析檢測細胞之倍增速率約11小時,在傳統流式細胞儀上分析結果也是相近的。在細胞轉染基因實驗中,我們利用CMOS 即時螢光細胞生長監控儀分析轉染成功細胞之平均螢光強度(mean fluorescence intensity, MFI) ,結果顯示轉染之細胞MFI比未轉染之細胞MFI提高,未來我們還能針對此儀器的濾片進行修正,用以增加顯著差異。因此,本研究結果證實此新穎儀器運用在即時觀察細胞增生與轉殖基因的表現,其功能與準確度與流式細胞儀的使用相較更具便捷優勢。

CMOS (Complementary Metal-Oxide-Semiconductor) based real-time fluorescent cells monitoring system can be applied for biological activity assay like measuring transfection efficiency, cell proliferation, reactive oxygen species (ROS) or cytotoxicity on novel drug test. The aim of our study is to confirm the feasibility and efficiency of our newly developed CMOS-based real-time fluorescent cells monitoring system for these biological assays. In cell proliferation study, we grew CFSE (carboxyfluorescein succinimidyl ester) -labeling RAW264.7 cells in vitro and the doubling time of these cells measured by our system approximately 11 hours. This result was in parallel with the data measured from conventional flow cytometry analysis. In gene transfer assay, we detected the expression of ectopic green fluorescence protein (GFP) in cells by mean of fluorescence intensity measured by CMOS system. The results showed that mean fluorescent intensity (MFI) of transfected cells was higher than that in untransfected cells. Currently, we confirmed that our innovative system was capable of performing real-time monitoring for cell growth and ectopic gene
expression in transfected cells. The functionality and accuracy of our system are competitive to flow cytometry.

Contents
中文摘要 ..........................................I
Abstract ....................................... II
謝誌 ............................................ IV
Contents ........................................ V
Figure of contents ............................. VII
Table of contents ............................... X
Chapter 1. Introduction ......................... 1
Chapter 2. Literature Review .................... 3
2.1 Comparison of CCD and CMOS....................... 3
2.2 Development of CMOS image system ................ 6
2.3 Evolution and principles of flow cytometry ..... 18
2.4 Detection of cell proliferation approaches with different dye .......................................22
2.4.1. PI (Propidium iodide)........................ 22
2.4.2. BrdU (Bromodeoxyuridine)..................... 23
2.4.3. CFSE (carboxyfluorescein succinimidyl ester) 24
2.5 Detection of reactive oxygen species induced cell apoptosis .......................................... 27
2.6 Analysis of cell transfection efficiency ....... 32
2.7 Aim ............................................ 33
Chapter 3. Materials and Methods ................... 34
3.1 Cells culture ............................................ 35
3.2 Procedure for Passaging Cells .................. 35
3.3 Cell labeling procedure with CFSE............... 35
3.4 Viable cell number counting .................... 36
3.5 Fluorescent microscope cell images analysis .... 36
3.6 Detection of mean fluorescence intensity (MFI) by flow cytometry ...........................................36
3.7 Detection of mean fluorescence intensity (MFI) by real-time fluorescence cell growth monitoring system......36
3.9 Statistical analysis ........................... 37
Chapter 4. Results ................................. 38
4.1 Cell proliferation assay ....................... 38
4.1.1. Using manual cell counting to calculate cell doubling time of RAW 264.7 cell labeled with 0.2μΜ CFSE ............................................... 38
4.1.2. Using fluorescent microscope to observe cell growth of RAW 264.7 cells labeled with 0.2 or 5 μΜ CFSE ... 41
4.1.3. Using flow cytometry to calculate cell doubling time of RAW 264.7 cell labeled with 0.2μΜ CFSE .......... 43
4.1.4. Using CMOS real-time fluorescence cell growth ..46
monitoring system to observe cell growth and calculate cell doubling time of RAW 264.7 cells labeled with 5 μΜ CFSE ..46
4.2 Cell transfection assay ........................ 50
4.2.1. Using fluorescent microscope images to observe transfection ..efficiency .......................... 50
4.2.1. Using flow cytometry to analyze transfection efficiency and MFI ..................................52
4.2.2. Using CMOS-based images to observe transfection efficiency and analyze MFI ......................... 53
Chapter 5. Discussion .............................. 56
Chapter 6. Conclusion... ........................... 58
Chapter 7. References .............................. 60
Information of Author .............................. 63
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