臺灣博碩士論文加值系統

癌細胞能逃脫免疫系統的監控，持續地在體內生長，主要是由於免疫系統無法辨識癌細胞及有效的產生抗腫瘤反應。利用免疫基因療法，以基因轉殖技術使癌細胞局部表現細胞激素，而激發抗腫瘤免疫反應是癌症治療的新趨勢。Fas/FasL系統參與CTL與NK細胞的細胞毒殺作用並維持免疫系統的恆定。研究顯示，使癌細胞大量表現FasL再注入老鼠體內，除可促使癌細胞死亡外，並可活化專一性抗腫瘤免疫力造成tumor regression。因此實驗中選用tetracycline- regulated retroviral gene expression system併用磷酸鈣或微脂體轉染的方法，使Lewis lung carcinoma (LLC1) cells在tetracycline的調控下大量表現FasL。以西方轉漬法及免疫螢光染色法測定出轉染後的LLC1細胞其細胞膜上具FasL表現，並以C57BL/6老鼠進行腫瘤生成特性分析，發現原生型LLC1細胞和vector control cell line皆能在老鼠體內產生腫瘤，而表現FasL的LLC1細胞則不具致癌性。從實驗結果推測FasL的表現會抑制腫瘤的生成，但卻不能有效的活化專一性抗腫瘤免疫力造成tumor regression。因此。若能結合其他治療性基因一同送入LLC1細胞內，或許可產生專一性抗腫瘤免疫反應促使tumor regression以達到免疫抗癌的目的。

The abilities of tumors to escape a functional immune system and persist in growth suggest that host immune mechanisms have failed to recognize and eliminate tumor cells. A variety of gene therapeutic strategies that induce systemic antitumor effects are under investigation, including the transfer of genes of cytokines to tumor cells. The Fas-Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression and stimulation of immune responses. It has been demonstrated that gene transfer of FasL on tumor cells inhibits tumors growth in vivo and generates potent antitumor immunity leading to regression of tumors. In this study, the Lewis lung carcinoma cells were transfected with recombinant retroviral vectors bearing human FasL gene (LLC1—FasL). This vector is an inducible expression vector, and FasL expression is regulated by tetracycline. LLC1—FasL cells expressed FasL on the cell surface was examined by Western blotting and immunostaining. FasL—transfacted LLC1 cells completely lost original tumorigenicity, but marked tumor regression was not expectedly observed after FasL gene transfer into LLC1 cells. These results indicate that although FasL—expressing LLC1 cells inhibited tumor growth, generation of a pivotal antitumor response likely depends on the synergistic combination of other therapeutic genes.

中文摘要………………………………………...…………….……..…..3
英文摘要………………………………………………………...…..…...4
壹、 緒論……………………………………………………………......5
一、Fas / FasL作用在免疫系統所扮演的角色……………………...5
二、Fas-FasL系統的應用…………..………………………..………11
三、研究動機與目的………………………………………….…….13
貳、 實驗材料……………………………………………...……...…..15
參、 實驗方法……………………………………………………..…..17
一、DNA定點突變（DNA mutagenesis）………………….……..17
二、質體的製備………………………………………………….….17
三、動物細胞培養及冷凍……………………………………….….18
四、質體DNA的轉染(Transfection)………………………….……19
五、生產病毒顆粒………………………………………….………..21
六、病毒的感染…………………………………………….……….21
七、RNA純化（RNA Purification）……………………….……….22
八、反轉錄作用（Reverse Transcription, RT）………………….…22
九、Luciferase活性測定…………………………………….………23
十、以Con A-sepharose抓取細胞膜上的FasL ……………...……..24
十一、西方墨法(Western blot)……..…………………….…………24
十二、免疫螢光標定法(Immunofluorescent labeling)……………...25
十三、細胞數測定（MTS assay）…………………………………25
十四、注射老鼠實驗……………………….……………………….25
肆、 結果……………………………………………………...……….27
一、製備表現FasL的LLC1細胞………………………….………27
二、LLC1 Tet-off 細胞株短暫地大量表現FasL之腫瘤發生性…30
三、植入大量表現FasL的LLC1細胞可使老鼠體內產生抗腫瘤免疫反應……………………………………………….…………31
伍、 討論………………………………………………………….…...33
一、FasL在其它cancer cell lines 的應用……………….…………33
二、Immunotherapy在LLC上的研究成果………………...………33
三、將FasL-expressing LLC1 cells植入老鼠皮下前若先以Trypsin處理細胞，會降低FasL 的antitumor activity………….……...34
四、FasL-expressing LLC1 cells，雖可引起發炎反應抑制腫瘤生長，但卻不能有效的產生具全身性的腫瘤專一性T細胞………..34
五、使用Tetracycline-regulated RevTet-off system的優缺點………35
陸、 參考文獻……………………………………………………..…..37

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