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研究生:張哲豪
研究生(外文):CHANG, CHE-HAO
論文名稱:應用細胞質譜儀定量細胞吞噬病毒粒子及細菌產出聚羥基烷酯聚合物分子的數量
論文名稱(外文):Quantitative measurement of Cellular Uptake of Viral particles and polyhydroxyalkanoates generated from Escherichia coli with Cell Mass Spectrometry
指導教授:彭文平彭文平引用關係
指導教授(外文):PENG, WEN-PING
學位類別:碩士
校院名稱:國立東華大學
系所名稱:物理學系
學門:自然科學學門
學類:物理學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
論文頁數:49
中文關鍵詞:細胞質譜儀
外文關鍵詞:Cell Mass Spectrometry
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目前癌症治療中,化學治療是最廣泛應用的治療方法,但傳統藥物投遞系統會有缺乏專一性或是對水溶解度不佳的總總限制,此限制會增加使用的藥物劑量而產生諸多的副作用,所以針對藥物投遞與導向式給藥系統,研究員做了很多的研究開發。本篇論文利用細胞質譜儀(CMS)研究細胞吞噬病毒奈米粒子後的質量變化,此研究目的跟近年來病毒奈米粒子的天然特性被應用當藥物載體有關。實驗結果顯示,使用類人類免疫缺陷病毒的慢病毒與人類急性淋巴細胞白血病T淋巴細胞一起培養,細胞的平均質量理論上勢必將會增加,但我們卻發現與理論不合的結果,也跟前人所做的結果會隨時間增加重量的結果不相吻合,我們懷疑是否細胞與病毒之間的表現並不是我們想像單向隨時間改變而改變,所以我們必須要再對細胞吞噬病毒是否產生質量的變化再做更進一步的確認與探討。另外,針對石化產品日漸普遍,塑膠產物無法分解,造成了環境的重大污染,聚羥基烷酯是一種由微生物於碳源過剩的環境中累積而成,其此高分子聚合物物理性質跟石化塑膠相同,但卻具有極高的生物相容性與可分解性的優點,所以我們嘗試將具有聚羥基烷酯的大腸桿菌放進細胞質譜儀中做量測,我們成功的運用細胞質譜儀系統量到腸桿菌的訊號,也成功得到了大腸桿菌與含有聚羥基烷酯的大腸桿菌質量差異分布圖,其產出質量差異大約是23%,因為大腸桿菌的尺寸過小所以量測上難度頗高,帶電量低很容易被電荷偵測器的基本雜訊給覆蓋,電荷偵測器基本雜訊為約1250顆電子,最好的訊號強度估計一顆大腸桿菌的帶電量約有3000~4000顆電子,這些都與之前2004年以離子阱質譜儀量測單顆細菌的質量量測結果相當符合;我們之後將嘗試對細胞質譜儀系統做出改善以提高量測效率,這對後續的聚羥基烷酯快速量測將有助益。
Nowadays, chemotherapy is widely used for the treatment of cancer. However, conventional drug delivery systems have some limitations, such as nonspecific targeting and lack of water solubility. It will need relatively large doses of drug and result in adverse side effects. Therefore, various drug delivery and drug targeting systems are under development. In recent years, viral nanoparticles are increasingly being used in drug carrier because of their natural property. Herein, we try to evaluate the cellular uptake of viral nanoparticles by cell mass spectrometry. The result shows that the mean-mass of CEM cells kept almost same after incubation with HIV-like lentiviruses at MOI(Mulitiplicity of Infection) 3. This might indicate the number of viral particles uptake by cells are not high enough to be detected. In addition, polyhydroxyalkanoates (PHA) are biocompatible and biodegradable polymers produced by Escherichia coli. Different PHA mono-molecule synthesized polymers are with its special thermal and mechanical properties. To detect the quantity of PHAs generated by Escherichia coli is time consuming. With cell mass spectrometry, we can detect the ion signal of single Escherichia coli particles tapped in ion trap. The measured mass is about 31010 Da which is the monomer mass of single Escherichia coli. Quantitative measurement of PHAs generated by Escherichia coli could be possible determined with cell mass spectrometry approach.



Keywords : cell mass spectrometry, drug carrier, viral nanoparticles, PHA, Escherichia coli

I. 摘 要 I
II. ABSTRACT II
III. 謝 辭 III
IV. 目 錄 IV
V. 圖目錄 VI
VI. 表目錄 VII
VII. 中英文縮寫對照表 VIII
VIII. 前 言 1
IX. 第二章 細胞質量質譜儀介紹 5
2-1 細胞質譜儀 (Cell Mass Spectrometer, CMS) 5
2-1-1 雷射導致超音波脫附法(Laser Induced Acoustic Desorption,LIAD) 6
2-1-2 四極離子阱(Quadrupole Ion Trap, QIT) 7
2-1-3 電荷偵測器(Charge Detector,CD) 8
2-1-4 訊號處理-正交小波包分解技術(Orthogonal Wavelet Packet Decomposition)與背景訊號降噪 9
X. 第三章 實驗樣品配製與實驗步驟 14
3-1 標準樣品配製 14
3-2 細胞培養與製備 15
3-2-1細胞株來源 15
3-2-2 細胞培養 15
3-2-3 細胞計數 15
3-2-4 病毒製作 16
3-2-5 病毒感染 16
3-3 細菌培養 16
3-4 CMS儀器樣品與實驗步驟 17
3-4-1儀器設備 17
3-4-2實驗步驟 17
XI. 第四章 實驗結果與討論 19
4-1 標準樣品(polystyrene)對CMS做校正之結果 19
4-1-1 細胞尺度量測之校正 19
4-1-2 細菌尺度量測之校正 22
4-2 CMS重覆測量單純CEM細胞的結果 23
4-3 量測CEM cells吞噬病毒的各個時間點 28
4-4 大腸桿菌的量測 33
XII. 第五章 結 論 35
XIII. 第六章 參考文獻 36

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