臺灣博碩士論文加值系統

免疫基因療法在癌症治療的應用上有很正面的潛力，本論文基於其中的優勢為建立一個具有良好調控特性的基因轉殖表現系統，將細胞間素-3（IL-3）轉殖進入腫瘤細胞並以藥物誘導表現，以期誘導表現的細胞間素-3能對腫瘤治療產生成效，並且降低其過量表現所造成的副作用。動物體外之實驗首先利用螢光蛋白質（EGFP）的誘導表現說明誘導性基因轉殖系統的功能完整。而後進行細胞間素-3的誘導表現系統建立，並於細胞間素-3之基因與蛋白質表現結果證實該系統的成功構築，再將此誘導性細胞間素-3基因轉殖腫瘤細胞注射入動物體內，建立腫瘤動物模式以觀察細胞間素-3之誘導表現與腫瘤治療的效果，但動物實驗結果發現新構築之腫瘤細胞，其腫瘤生成率極低，無法在動物中形成腫瘤，自然無法觀察比較其治療成效。檢測動物血清中細胞激素-3的含量以及動物脾臟腫大觀察也同樣沒有正面的結果說明細胞間素-3被誘導表現的證據。推測此轉殖系統的建構流程冗長不利於實驗中所使用之TRAMP腫瘤細胞，建議可改用其他腫瘤細胞與動物模式，或改以腺病毒轉染方式為媒介，簡化流程並得到較高的轉殖效率。

目錄
英文摘要.................................................Ⅰ
中文摘要.................................................Ⅱ
誌謝.....................................................Ⅲ
目錄.....................................................Ⅳ
第一章 緒論
1.1 腫瘤的免疫基因治療
1.1.1 癌症的傳統治療方式.........................1
1.1.2 癌症的基因治療.............................3
1.1.3 癌症的免疫基因治療.........................4
1.1.4 癌症免疫基因治療中常用的細胞激素及其作用機制..........................................5
1.2 轉殖基因的條件式調控
1.2.1 傳統轉殖基因系統的限制.....................6
1.2.2 調節性轉殖基因系統的需求...................7
1.2.3 條件式調控轉殖基因系統-Tet system的建立....8
1.3 細胞間素-3的功能及對腫瘤的影響
1.3.1 細胞間素-3的免疫與造血功能...............10
1.3.2 細胞間素-3於腫瘤基因治療的免疫功能.......11
1.4 研究目的與內容................................13
第二章 材料與方法
2.1 細胞株與細胞培養..............................14
2.2 質體的構築
2.2.1 勝任細胞的製備............................14
2.2.2 細菌熱休克轉化作用........................15
2.2.3 質體的製備................................15
2.2.4 限制酵素切割反應..........................16
2.2.5 DNA片段的電泳分離.........................16
2.2.6 DNA片段的純化.............................17
2.2.7 聚合酵素連鎖反應..........................17
2.2.8 DNA接合反應...............................18
2.2.9 Quick Screening............................19
2.3 誘導性調控基因在細胞中的表現
2.3.1 細胞的轉染作用............................20
2.3.2 誘導性調控基因轉殖細胞的建立..............20
2.3.3 細胞對抗生素藥物的敏感度測試..............21
2.3.4 細胞存活試驗..............................22
2.3.5 基因轉殖細胞的調控藥物處理...............22
2.3.6 螢光蛋白質的顯微鏡觀察...................23
2.3.7 細胞RNA的萃取...........................24
2.3.8 RT-PCR...................................24
2.3.9 酵素連結免疫吸附法.......................25
2.4 動物實驗
2.4.1 實驗動物.................................27
2.4.2 皮下注射.................................27
2.4.3 基因調控藥物處理.........................28
2.4.4 血清採樣.................................28
2.4.5 腫瘤切片組織.............................28
第三章 實驗結果
3.1 誘導性細胞間素-3基因轉殖腫瘤細胞的建立
3.1.1 構築細胞間素-3質體（pTRE2-IL3）........29
3.1.2 螢光蛋白質質體的序列確認................30
3.1.3 TRAMP細胞對抗生素藥物G418以及Hygromycin的敏感度測試..............................31
3.1.4 誘導性調控基因（Tet-On）轉殖細胞的建立..32
3.1.5 誘導性螢光蛋白質基因轉殖細胞的建立與表現......................................32
3.2 誘導性調控細胞間素-3基因在細胞層次中的表現
3.2.1 IL-3於細胞中的mRNA表現................33
3.2.2 IL-3於細胞中的蛋白質表現...............34
3.3 誘導性調控細胞間素-3腫瘤細胞（TT-IL3）在活體動物中的表現與影響
3.3.1 TT-IL3在動物體中的腫瘤生成.............35
3.3.2 IL-3蛋白質於動物血清中的檢測...........36
3.3.3 實驗動物的脾腫大觀察....................37
第四章 討論.............................................38
第五章 參考文獻.........................................44

第五章 參考文獻
1. Nasu, Y., et al., Suicide gene therapy for urogenital cancer: current outcome and prospects. Mol Urol, 2000. 4(2): p. 67-71.
2. Frank, D.K., et al., Bystander effect in the adenovirus-mediated wild-type p53 gene therapy model of human squamous cell carcinoma of the head and neck. Clin Cancer Res, 1998. 4(10): p. 2521-8.
3. Hamblin, A.S., Cytokines and cytokine receptors. 1993, New York: Oxford.
4. Hock, H., et al., Mechanisms of rejection induced by tumor cell-targeted gene transfer of interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or interferon gamma. Proc Natl Acad Sci U S A, 1993. 90(7): p. 2774-8.
5. Parmiani, G., M. Rodolfo, and C. Melani, Immunological gene therapy with ex vivo gene-modified tumor cells: a critique and a reappraisal. Hum Gene Ther, 2000. 11(9): p. 1269-75.
6. Wong, P.M., et al., Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder. Mol Cell Biol, 1989. 9(2): p. 798-808.
7. Pulaski, B.A., et al., Interleukin 3 enhances development of tumor-reactive cytotoxic cells by a CD4-dependent mechanism. Cancer Res, 1993. 53(9): p. 2112-7.
8. Hynes, N.E., et al., Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells. Proc Natl Acad Sci U S A, 1981. 78(4): p. 2038-42.
9. Mayo, K.E., R. Warren, and R.D. Palmiter, The mouse metallothionein-I gene is transcriptionally regulated by cadmium following transfection into human or mouse cells. Cell, 1982. 29(1): p. 99-108.
10. Wurm, F.M., K.A. Gwinn, and R.E. Kingston, Inducible overproduction of the mouse c-myc protein in mammalian cells. Proc Natl Acad Sci U S A, 1986. 83(15): p. 5414-8.
11. Triezenberg, S.J., R.C. Kingsbury, and S.L. McKnight, Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev, 1988. 2(6): p. 718-29.
12. Flint, J. and T. Shenk, Viral transactivating proteins. Annu Rev Genet, 1997. 31: p. 177-212.
13. Babb, R., et al., DNA recognition by the herpes simplex virus transactivator VP16: a novel DNA-binding structure. Mol Cell Biol, 2001. 21(14): p. 4700-12.
14. Gossen, M. and H. Bujard, Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A, 1992. 89(12): p. 5547-51.
15. Gossen, M., et al., Transcriptional activation by tetracyclines in mammalian cells. Science, 1995. 268(5218): p. 1766-9.
16. Lantz, C.S., et al., Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature, 1998. 392(6671): p. 90-3.
17. Frendl, G., Interleukin 3: from colony-stimulating factor to pluripotent immunoregulatory cytokine. Int J Immunopharmacol, 1992. 14(3): p. 421-30.
18. Yeh, K.Y., et al., IL-3 enhances both presentation of exogenous particulate antigen in association with class I major histocompatibility antigen and generation of primary tumor-specific cytolytic T lymphocytes. J Immunol, 1998. 160(12): p. 5773-80.
19. McBride, W.H., G. J. Dougherty, A. E. Wallis, J. S. Economou, and C. S. Chiang, Interleukin-3 in gene therapy of cancer. Folia Biologica, 1994. 40: p. 62.
20. Chiang, C.S., et al., Effects of IL-3 gene expression on tumor response to irradiation in vitro and in vivo. Cancer Res, 1997. 57(18): p. 3899-903.
21. Fung, M.C., et al., Molecular cloning of cDNA for murine interleukin-3. Nature, 1984. 307(5948): p. 233-7.
22. Tet-Off and Tet-On Gene Expresion Systems User Manual. 1999: CLONTECH.
23. Benihoud, K., P. Yeh, and M. Perricaudet, Adenovirus vectors for gene delivery. Curr Opin Biotechnol, 1999. 10(5): p. 440-7.
24. Kovesdi I, Douglas E. B., Bruder J. T., and Wickham T.J., Adenoviral vectors for gene tranfer. Current Opinion in Biotechnology, 1997. 8: p. 583-589.
25. Mizuguchi, H. and T. Hayakawa, Characteristics of adenovirus-mediated tetracycline-controllable expression system. Biochim Biophys Acta, 2001. 1568(1): p. 21-9.
26. Molin, M., et al., Two novel adenovirus vector systems permitting regulated protein expression in gene transfer experiments. J Virol, 1998. 72(10): p. 8358-61.