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研究生:伍婉琳
論文名稱:利用四環黴素調控反轉錄病毒表現FAsL於Lewis肺癌細胞產生免疫治療
論文名稱(外文):Genetic immunotherapy of eatablished lewis lung carcinoma with fasl expressed by tetracycline-regulated retroviral vector
指導教授:唐世杰
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:47
中文關鍵詞:免疫治療FasL四環黴素調控
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中文摘要
惡性腫瘤是現今嚴重的疾病之一,其療法包括化學療法、放射性療法和外科手術,但治癒率與存活率皆低,至今仍無一有效方式能專一且不傷害正常細胞下去除腫瘤。癌細胞藉insufficient costimulation、tolerancec或immunosuppression方式使免疫系統無法辨認癌細胞抗原,而躲避免疫系統攻擊,不斷增生。基因療法利用基因轉殖的技術改變癌細胞的生理特性,使免疫系統能辨別除可消滅定位腫瘤,也可消除轉移的癌細胞。Fas/FasL參與維持免疫系統恆定和對抗病毒及癌細胞。研究顯示使癌細胞大量表現FasL後,植入體內,除被修飾的癌細胞死亡外,再植入或已轉移的癌細胞皆egression。
實驗中選用致癌性高,且易產生轉移的Lewis lung carcunoma (LLC1)作為實驗細胞。選擇Rev Tet-off表現系統,可藉tetracyclin調控FasL的表現。此系統利用retrovirus進行基因轉殖,其基因轉殖效率高且只能插入具分裂能力的細胞,具活體利用價值。
選殖大量表現FasL的LLC1細胞株,再植入老鼠體內可發現其具有抑制腫瘤生長的作用,由此可知,FasL具有治療惡性腫瘤的潛力。結合其他具抗腫瘤免疫力的蛋白質表現例如:interferon、IL-2或IL-12以促進體內免疫系統活化,達到癌症治療的目的。

目錄
中文摘要
英文摘要
壹、序論……………………………………………….……………1
一、 Fas/FasL的簡介………………………………………..2
二、 Immunoregulatory functions of the Fas/FasL system…………………………………………………………7
三、 FasL in Immune privilege sites………………….…….9
四、 FasL in transplantation………………………..………10
五、 FasL in inflammation………………………………….11
六、 FasL in tumor cells………………………………...…….13
七、 實驗動機與目的………………………...……………14
貳、實驗材料………………………………………………….……16
參、實驗方法…………………………………………………..………19
一、 質體DNA定點突變………………………...…………..19
二、Plasmid construction…………………………………………19
三、動物細胞培養及冷凍………………………………………..20
四、質體DNA的轉染…………………………..………………21
五、生產病毒顆粒……………………………….………………23
六、病毒的感染……………………………………….…………23
七、RNA純化………………………………………...…………..24
八、反轉錄作用………………………………………………….24
九、Luciferase活性測定…………………………………...…….25
十、FasL表現的測定……………………………….……………26
十一、注射老鼠實驗……………………………………………..27
肆、結果………………………………………………….…………28
一、Rev-Tet-off表現系統…………………………………….28
二、血清中含有其他因子可促進CMV promoter的活性…..…29
三、建立可受tetracycline 調節表現FasL的LLC1細胞株….....30
四、Animal experiment…………….……………………………32
伍、討論…………………………………………….…………………34
一、Rev Tet-off 表現系統的優缺點………….………………….34
二、Immunotherapy在LLC上的研究成果……………….……..35
三、LLC1不表現Fas,可誘導發炎反應………………..………..36
四、大量表現FasL抑制腫瘤生長但無法誘發全身性免疫力…36
五、結合其他蛋白質表現,以利誘導抗腫瘤免疫力………..38
陸、參考文獻……………………………………………...………..40

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