(3.235.25.169) 您好!臺灣時間:2021/04/20 17:22
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:楊湘梅
研究生(外文):Yang, Hsiang-Mei
論文名稱:大腸桿菌胞嘧啶去胺�t在基因治療之應用
論文名稱(外文):The Feasibility of Using Bacterial Cytosine Deaminase Gene as a Positive or Negative Selection Marker in the Retroviral Vector for Gene Therapy
指導教授:吳昭良
指導教授(外文):Wu Chao-Liang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1996
畢業學年度:84
語文別:中文
論文頁數:62
中文關鍵詞:胞嘧啶去胺反轉錄病毒載體正向篩選負向篩選
外文關鍵詞:cytosine deaminaseretroviral vectorpositive selectionnegative selection
相關次數:
  • 被引用被引用:2
  • 點閱點閱:125
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
近幾年來,反轉錄病毒載體系統已發展成為一重要的基因傳送工具,
但考量整個系統,仍有一些限制或缺點。我們的實驗即是要提出可行的策
略,來提高系統應用的安全性及適用範圍。選用大腸桿菌胞嘧啶去胺(
Escherichia coli cytosine deaminase;CD)基因作為反轉錄病毒載體上
的負向篩選標記,使之在基因傳送過程中,連同其它功能性基因一起被送
入目標細胞中,一旦轉型後的細胞發生不如預期的效果,則可用
5-fluorocytosine(5FC) 加以處理,對於那些可表現CD活性的轉型細胞,
能將無害的5FC代謝成具細胞毒性的 5-fluorouracil(5FU),故可選擇性
地殺死那些細胞。配合自殺基因的應用,使人們在完成基因傳送後,還能
掌握那些含有外來基因的細胞,隨時視實際狀況的變化,決定細胞的生死
。實驗進行,首先,構築一同時具有CD及抗neomycin基因的反轉錄病毒載
體,然後利用電破法將之送入包裝細胞,挑選抗G418的細胞株,培養7
至14天後,收集上清液,測病毒效價,效價最高可達 10cfu/mL 同時經
由對5FC的細胞毒性測試,得知可表現CD的細胞株,對5FC有較高之敏感性
。更進一步以病毒去感染老鼠膀胱癌細胞MBT2,將此轉型細胞打入老鼠體
內,模擬一個癌化的轉型細胞存在活體內的狀況,再腹腔注射5FC,以期
能夠選擇性地殺死含有CD基因的細胞。另外,我們利用一個新策略,使CD
發揮正向篩選的功能,以取代neo基因,進而增加了載體對外來基因大小
的容許度。主要是在培養基中,同時添加cytosine及5FU,對於可表現CD
的細胞株,能將 cytosine 去胺代謝成 uracil,uracil 可與5FU競爭,
減低後者對細胞酵素的抑制機會,而提高細胞存活率。實驗結果顯示確時
可達良好的正向篩選效果。
Retroviral gene therapy is a technique that allows tansfer
of genes into a broad host range with high efficiency. The risk
of unpredicatable insertional mutagenesis is the major concern
in all gene transfer with random integration. To minimize the
uncertainty, we constructed a retroviral vector with positive
and negative selection modules. The enzyme cytosine deaminase
(CD), absent in mammals, catalyzes the deamination of cytosine
to uracil. It can also convert the innocuous compound
5-fluorocytosine to a highly toxic compound 5-fluorouracil,
which has been proved lethal to the cells and is widely used in
cancer chemotherapy. The cells expressing the bacterial gene for
cytosine deaminase would be eliminated in the presence of
5-fluorocytosine. Combined with neomycin phosphotransferase
(neo) as the positive selection module, a retroviral vector
containing neo and CD genes has been constructed. The cells
infected by this vector show higher susceptibility to
5-fluorocytosine compared with those infected by their control
counterpart. The lower tumor incidence has also be observed in
the animal experiments which simulated the unpleasant occurrence
of tumor after gene transfer. Furthermore, to provide more space
for accommodation of foreign cDNA, a retroviral vector
accompanied with a novel positive selection scheme for the
existence of bacterial CD has been established. The CD gene
could serve not only as positive but also as negative selection
modules for cells transfected by retroviral vector containing CD
gene itself. This strategy might facilitate the possibility to
control the cells with retroviral infections after furnishing
their effect of the transgene either in vivo or ex vivo.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔