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研究生:徐菁穗
研究生(外文):Ching-Suei Hsu
論文名稱:桿狀病毒/哺乳動物細胞表現系統轉導條件最適化之研究
指導教授:胡育誠胡育誠引用關係
指導教授(外文):Yu-Chen Hu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:40
中文關鍵詞:桿狀病毒轉導基因傳遞哺乳動物細胞蛋白質表現
外文關鍵詞:baculovirus transductiongene deliverymammalian cellprotein expression
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雖然目前已有許多研究報導以桿狀病毒作為載體可將基因送入哺乳動物細胞,然而研究多偏向以桿狀病毒作為基因治療載體的應用,對於最佳的轉導條件則未有系統性的研究。本研究中我們提出使用未濃縮的桿狀病毒將基因送入HeLa細胞株的轉導方法。在轉導時我們取出培養基後將細胞加入病毒液,以磷酸緩衝溶液(D-PBS)作為轉導環境溶液於室溫下進行轉導4 h,可表現報導基因並達到75-85 %的轉導效率。相較於其他研究團隊所提出將細胞與濃縮的病毒以培養基為環境溶液,於37 °C轉導1 h的方法,本研究提供的方法既簡單又有效率。經由以上最適化轉導條件研究我們發現:(1)將高劑量的病毒與HeLa細胞株進行轉導至少4 h可得到良好的轉導效率;(2)於37 °C下,桿狀病毒的半生期會明顯地降低;(3)當細胞生長到多層時,使用EGTA前處理無法明顯地增加感染效率;(4)相較於TNM-FH與D-PBS,DMEM不適合作為轉導時的環境溶液,因此我們推測DMEM中含有抑制轉導效率的因子。關於DMEM造成低轉導效率的原因,本研究中測試了數種可能的因素,如pH、陽離子效應及glucose濃度等,目前仍未發現DMEM中影響轉導效率的因素;(5)使用本研究中所提出的轉導方法雖然與一般細胞培養及利用病毒轉導的條件相差甚大,對細胞生長只有短暫的影響,對細胞的存活率沒有明顯的影響,也不會出現細胞毒性。本研究提出了省略濃縮步驟時,桿狀病毒/哺乳動物細胞表現系統的最佳轉導策略,經由此策略可成功地將重組基因送入哺乳動物細胞中,並得到與其他研究者相似的轉導結果。經由這樣的研究結果,成功的避免了超高速離心可能對病毒活性造成的影響及大量生產時超高速離心程序帶來的不便。
Although baculovirus-mediated gene delivery into mammalian cells has been reported in many literatures, previous studies were focused on the application of baculovirus as a gene therapy vector. Besides, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus was proposed for simple and efficient gene delivery into HeLa cell. We found that �l75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25�aC using Dulbecco’s phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasted to previous protocols in which transduction occurs for 1 h at 37�aC using the growth medium (e.g. DMEM) as the surrounding solution. Investigation of the physical parameters led to the finding that (1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; (2) the half-life of baculovirus dramatically decreased at 37�aC; (3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; (4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction; several factors (e.g. pH, ions, and glucose concentration) were tested to investigate the low efficiency caused by DMEM, but the reasons were not clear yet; (5) the transduction conditions we proposed was totally different from the usual culture conditions and the transduction conditions used by other groups. However, this protocol did not cause cytopathic effect but only transient effect on grow curve. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eucaryotic protein production in a larger scale.
中文摘要.....II
英文摘要.....III
目錄.........IV
圖表目錄.....VI
第一章 緒論.............................................1
第二章 文獻回顧.........................................2
2.1. 桿狀病毒與昆蟲細胞.............................2
2.2. 桿狀病毒表現載體系統的發展與應用...............3
2.3. 桿狀病毒表現載體進入細胞之路徑.................4
2.4. 桿狀病毒/哺乳動物細胞表現系統的發展............6
2.5. 研究動機.......................................7
第三章 實驗材料及分析方法...............................8
3.1. 哺乳動物細胞與培養基...........................8
3.2. 昆蟲細胞與培養基...............................8
3.3. 重組桿狀病毒載體...............................8
3.4. 病毒量的放大與濃縮.............................8
3.5. 桿狀病毒轉導哺乳動物細胞.......................9
3.6. 分析方法.......................................9
3.6.1. 細胞計數及存活率
( Trypan blue dye exclusion method )...........9
3.6.2. 終點稀釋法( End-point dilution method ).......10
3.6.3. 轉導比例、平均螢光強度及總螢光強度定量........11
第四章 結果與討論......................................12
4.1. 不同病毒劑量與轉導時間對螢光蛋白產量的影響....12
4.2.1. 轉導溫度對螢光蛋白表現的影響..................14
4.2.2. 溫度對病毒活性的影響..........................15
4.3. 不同轉導環境對螢光蛋白表現的影響..............16
4.3.1 轉導環境溶液對病毒活性的影響..................17
4.3.2 pH對螢光蛋白表現的影響........................17
4.3.3 離子對螢光蛋白表現的影響......................18
4.3.4 Glucose濃度對螢光蛋白表現的影響...............18
4.4. 轉導程序對細胞生長的影響......................19
4.5. 使用EGTA前處理對螢光蛋白表現的影響............20
第五章 結論............................................36
參考文獻...............................................39

圖表目錄
圖目錄
圖2-1桿狀病毒的分類.....................................2
圖2-2桿狀病毒進入昆蟲細胞的機制.........................5
圖4-1依一般方式轉導之螢光表現圖........................23
圖4-2病毒劑量對轉導效率的影響..........................23
圖4-3病毒劑量、轉導時間 (A)與發螢光細胞比例的關係圖
(B)與平均螢光強度關係圖...........................24
圖4-4隨轉導時間增加病毒進入細胞的關係圖................25
圖4-5不同轉導溫度 (A)與發螢光細胞比例的關係圖
(B)與平均螢光強度的關係圖.........................26
圖4-6溫度對病毒活性的影響..............................27
圖4-7不同環境溶液 (A)與發螢光細胞比例的關係圖
(B)與平均螢光強度的關係圖.........................28
圖4-8不同環境溶液對病毒活性的影響......................29
圖4-9環境溶液中不同pH值 (A)與發螢光細胞比例的關係圖
(B)與平均螢光強度的關係圖.........................30
圖4-10環境溶液中離子 (A)與發螢光細胞比例的關係圖
(B)與平均螢光強度的關係圖。.......................32
圖4-11環境溶液中glucose濃度 (A)與發螢光細胞比例的關係圖
(B)與平均螢光強度的關係圖.........................33
圖4-12轉導程序對細胞生長的影響。(A)轉導後細胞的生長速率
(B)轉導後細胞的存活率.............................34
圖4-13 EGTA前處理 (A)與發螢光細胞比例的關係圖
(B)與平均螢光強度的關係圖.........................35
表目錄
表4-1桿狀病毒在不同溫度下的半生期......................27
表4-2比較實驗中所使用的環境溶液與一般情形下哺乳動物細胞內外 的陽離子濃度...........................................31
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