臺灣博碩士論文加值系統

摘要
本實驗的目的評估結合PSA攝護腺特異性抗原與細胞激素GM-CSF之DNA疫苗在治療攝護腺癌的可行性。首先建立一個可同時表現人類PSA基因與老鼠GMCSF基因的DNA疫苗，再來我們將此質體DNA注射到C57/B6J與C3H/HeN兩種品系的老鼠體內比較不同的品系引起免疫反應，並與單獨表現人類PSA基因的DNA疫苗所引起的免疫反應作比較來評估。
結果發現此DNA疫苗在兩種品系老鼠體內並未引起體液性免疫反應，在血清中測得因肌肉注射引起的IgG1與IgG2a並沒有顯著差異。以西方墨點法也無法從血清中測得anti-PSA抗體的存在，然而從注射過疫苗的老鼠體內取出的脾臟純化出T細胞所做的毒殺型T細胞試驗可看出毒殺Nfsa-PSA的效果。但是兩種品系的老鼠皆可觀察到相同的毒殺效果，因此可能為殺手細胞所引起的非專一型的毒殺。
此構築的PSA之DNA疫苗在引起特異性免疫作用方面為失敗的，原因是我們只能以RT-PCR證實NFsa-PSA在mRNA有表現PSA，但不論在上清液若細胞溶解後的蛋白質中皆無法以ELISA測到。皆無法測得其存在。這可能是因我們所構築的PSA中的點突變或缺少PSA基因非轉譯的區域所造成。要解決此一問題，得以序列完全相同的PSA基因才行。另外使用PSA的轉殖基因鼠或使用老鼠本身的攝護腺抗原probasin與GM-CSF結合當作疫苗為建議改善的方法。

Abstract
The aim of this study is to assess the feasibility of treating prostate cancer with the DNA vaccine containing PSA and GM-CSF genes. First, we cloned a DNA plasmid to express the gene of human PSA and the gene of mice GM-CSF simultaneously. Second, we injected this plasmid into C57BL/6J and C3H/HeN mice to evaluate the immunity of different strains of mice. The effect of GM-CSF on DNA vaccine was evaluated by comparing its immunity with the immunity generated by PSA alone.
The results of this study showed that constructed plasmid could not generate humoral immunity against PSA in both strains of mice. There was not difference in IgG1 or IgG2a level following intramuscular DNA vaccine. Western blot was unable to detect the anti-PSA antibody in the serum. However, the CTL assay showed the cytotoxicity of spleen cells from immuned-mice against NFsa-PSA cells. The CTL responses were observed in both strains of mice, which indicated that this response is likely due to the non-specific killing of NK-cells.
The failure of the generation of specific immunity following DNA vaccine could be the results of PSA cloning. Because we found that Nfsa-PSA cells could only express mRNA of PSA as assessed by RT-PCR. We were unable to detect hPSA protein in both the supernatant and cell lysis by ELISA assay. We are currently unaware whether this effect is due to the point mutations of PSA that we cloned or the lack of non-translated regions of PSA gene. A clone of without mutation could be one way to answer this question. Beside, to improve this model, the use of PSA transgenic mice or probasin antigen, which is the prostate specific-antigen of the mouse, is suggested.

目錄
中文摘要……………………………………………………….I
英文摘要……………………………………………………...III
誌謝…………………………………………………………....V
目錄…………………………………………………………...VI
第一章、 前言
1.1 攝護腺癌……………………………………………………1
1.2 癌症免疫治療………………………………………………2
1.2.1 DNA疫苗…………………….…………………....4
1.3 攝護腺癌發展中的免疫治療……………………………....6
1.4 攝護腺特異性抗原…………………….…………………...7
1.5 顆粒球巨噬細胞群落刺激因子….………………………...9
1.6 研究目的與內容…………………………………………..11
第二章、 材料與方法
2.1 細胞培養………………………………..………… ……...12
2.2 質體的構築.…………………………………………….…12
2.2.1 勝任細胞（Competent Cell）的製備…….….….12
2.2.2 細菌熱休克轉殖法（Heat-Shock Transformation）……………………………….....13
2.2.3 質體的製備DNA……..…………………….……...14
2.2.3.1 微量DNA製備（miniprep）………..………...14
2.2.3.2 中量製備DNA（midiprep）.…….…………...14
2.2.3.3 大量製備DNA（maxiprep）……………….15
2.2.4 RNA的純化（Total RNA Isolation）……….…….16
2.2.5 RT-PCR…………………….………………….…17
2.2.5.1 反轉錄作用 (Reverse Transcription).………17
2.2.5.2 聚合酵素連鎖反應……………………...…..18
2.2.6 洋菜膠電泳……………………………….……...18
2.2.7 DNA片段的分離（Gel Extraction）……….…..19
2.2.8 限制酵素切割反應…………….………….……..19
2.2.9 DNA的純化（Purification）.……………………...20
2.2.10 DNA片段的去磷酸化（de-phosphoylation）.……20
2.2.11 DNA的接和（Ligation）………………..………...21
2.2.12 聚合酵素連鎖反應…………………………..…..21
2.3 在轉染細胞中的表現…………………………………………22
2.3.1 細胞對Puromycin的敏感性試驗I…………...…22
2.3.2 細胞存活試驗 (MTT assay)………………...…..23
2.3.3 細胞對Puromycin的敏感性試驗II…………......23
2.3.4 細胞的轉染 (Transfection)…………………..….24
2.3.5 酵素連結免疫吸附法……………………………24
2.3.5.1 GMCSF ELISA……………………………...24
2.2.5.2 PSA ELISA kit…………………………...….26
2.4 在動物體內的表現………………………………………...….26
2.4.1 實驗動物…………………………………………26
2.4.2 肌肉注射…………………………………………26
2.4.3 血清的抽取………………………………………27
2.4.4 酵素連結免疫吸附法……………………………27
2.4.4.1 IgG1、IgG2a ELISA……………..…………27
2.4.4.2 Anti-PSA ELISA ……………………………28
2.4.5 西方墨點法（Western blot）………………………29
2.4.5.1 細胞的溶解………………………………….29
2.4.5.2 製備電泳膠………………………………….29
2.4.5.3 免疫轉印…………………….………………30
2.4.6 T細胞的純化…………………………………….30
2.4.6.1 備製尼龍管柱（nylon column）……………30
2.4.6.2 細胞處理…………………………………….31
2.4.6.3 漲破血球…………………………………….31
2.4.6.4 純化T細胞………………………………….31
2.4.7 毒殺型T細胞試驗（CTL assay）………………32
第三章、 結果
3.1 構築IRES-PSA-GMCSF之DNA疫苗……………………….33
3.1.1 構築IRES-PSA-GMCSF之質體DNA…………33
3.2 注射IRES-PSA-GMCSF與IRESp-PSA到C57/B6J 和C3H/HeN兩種品系中，並測試所引起的免疫反應…………35
3.2.1 施打DNA疫苗並採取血清…………………….35
3.2.2 血清中IgG1之ELISA結果……………………36
3.2.3 血清中IgG2a之ELISA結果…………………….37
3.2.4 血清中PSA之ELISA結果…………………….39
3.2.5 血清中GMCSF之ELISA結果………………….39
3.2.6 西方墨點法測血清中Anti-PSA抗體之結果…...39
3.3 建立 Nfsa-PSA細胞…………………………………………..40
3.3.1 細胞對Puromycin的敏感性試驗I……………...41
3.3.2 細胞對Puromycin的敏感性試驗II…………….41
3.3.3 轉染細胞（Nfsa-PSA）中人類PSA的mRNA表現………………………………………………….41
3.3.4 在轉染細胞中（Nfsa-PSA）中人類PSA蛋白質的表現……………………………………………….42
3.4 注射DNA疫苗的老鼠之毒殺型T細胞試驗（CTL assay）果………………………………………………………44
第四章、討論…………………………………………………………46
第五章、參考文獻……………………………………………54
圖表……………………………………………………………58
附錄一…………………………………………………………78
附錄二…………………………………………………………79

1. Rosenberg, S.A., Immunotherapy and gene therapy of cancer. Cancer Res, 1991. 51(18 Suppl): p. 5074s-5079s.
2. Hoover, H.C., Jr., et al., Prospectively randomized trial of adjuvant active-specific immunotherapy for human colorectal cancer. Cancer, 1985. 55(6): p. 1236-43.
3. Fearon, E.R., et al., Induction in a murine tumor of immunogenic tumor variants by transfection with a foreign gene. Cancer Res, 1988. 48(11): p. 2975-80.
4. Gansbacher, B., et al., Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity. J Exp Med, 1990. 172(4): p. 1217-24.
5. Curti, B.D., et al., Interstitial pressure of subcutaneous nodules in melanoma and lymphoma patients: changes during treatment. Cancer Res, 1993. 53(10 Suppl): p. 2204-7.
6. Akiyoshi, T., [Cancer vaccine therapy using peptides derived from tumor-rejection antigens]. Gan To Kagaku Ryoho, 1997. 24(5): p. 511-9.
7. Saenz-Badillos, J., S.P. Amin, and R.D. Granstein, RNA as a tumor vaccine: a review of the literature. Exp Dermatol, 2001. 10(3): p. 143-54.
8. Schumacher, K., Keyhole limpet hemocyanin (KLH) conjugate vaccines as novel therapeutic tools in malignant disorders. J Cancer Res Clin Oncol, 2001. 127 Suppl 2: p. R1-2.
9. Dummer, R., et al., Immune stimulatory potential of B7.1 and B7.2 retrovirally transduced melanoma cells: suppression by interleukin 10. Br J Cancer, 1998. 77(9): p. 1413-9.
10. Donnelly, J.J., et al., DNA vaccines. Annu Rev Immunol, 1997. 15: p. 617-48.
11. Gurunathan, S., D.M. Klinman, and R.A. Seder, DNA vaccines: immunology, application, and optimization*. Annu Rev Immunol, 2000. 18: p. 927-74.
12. Chow, Y.H., et al., Development of Th1 and Th2 populations and the nature of immune responses to hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes. J Immunol, 1998. 160(3): p. 1320-9.
13. Haupt, K., M. Roggendorf, and K. Mann, The potential of DNA vaccination against tumor-associated antigens for antitumor therapy. Exp Biol Med (Maywood), 2002. 227(4): p. 227-37.
14. Kuwabara, H., H. Uda, and I. Takenaka, Immunohistochemical detection of sialosyl-Tn antigen in carcinoma of the prostate. Br J Urol, 1997. 80(3): p. 456-9.
15. Murphy, G.P., et al., Use of artificial neural networks in evaluating prognostic factors determining the response to dendritic cells pulsed with PSMA peptides in prostate cancer patients. Prostate, 2000. 42(1): p. 67-72.
16. Slovin, S.F., et al., Carbohydrate vaccines in cancer: immunogenicity of a fully synthetic globo H hexasaccharide conjugate in man. Proc Natl Acad Sci U S A, 1999. 96(10): p. 5710-5.
17. Zier, K.S. and B. Gansbacher, Tumour cell vaccines that secrete interleukin-2 (IL-2) and interferon gamma (IFN gamma) are recognised by T cells while resisting destruction by natural killer (NK) cells. Eur J Cancer, 1996. 32A(8): p. 1408-12.
18. Lodge, P.A., et al., Dendritic cell-based immunotherapy of prostate cancer: immune monitoring of a phase II clinical trial. Cancer Res, 2000. 60(4): p. 829-33.
19. Bowden, C.J., et al., A phase I/II study of continuous infusion suramin in patients with hormone-refractory prostate cancer: toxicity and response. Cancer Chemother Pharmacol, 1996. 39(1-2): p. 1-8.
20. Ablin, R.J., A retrospective and prospective overview of prostate-specific antigen. J Cancer Res Clin Oncol, 1997. 123(11-12): p. 583-94.
21. Eder, J.P., et al., A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer. Clin Cancer Res, 2000. 6(5): p. 1632-8.
22. Small, E.J., et al., Therapy of advanced prostate cancer with granulocyte macrophage colony-stimulating factor. Clin Cancer Res, 1999. 5(7): p. 1738-44.
23. Hung, K., et al., The central role of CD4(+) T cells in the antitumor immune response. J Exp Med, 1998. 188(12): p. 2357-68.
24. Dranoff, G., et al., Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A, 1993. 90(8): p. 3539-43.
25. Liu, H.M., et al., Immunostimulatory CpG oligodeoxynucleotides enhance the immune response to vaccine strategies involving granulocyte-macrophage colony-stimulating factor. Blood, 1998. 92(10): p. 3730-6.
26. Sin, J.I., et al., Enhancement of protective humoral (Th2) and cell-mediated (Th1) immune responses against herpes simplex virus-2 through co-delivery of granulocyte-macrophage colony-stimulating factor expression cassettes. Eur J Immunol, 1998. 28(11): p. 3530-40.
27. Rissoan, M.C., et al., Reciprocal control of T helper cell and dendritic cell differentiation. Science, 1999. 283(5405): p. 1183-6.
28. Pulendran, B., et al., Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc Natl Acad Sci U S A, 1999. 96(3): p. 1036-41.
29. Kusakabe, K., et al., The timing of GM-CSF expression plasmid administration influences the Th1/Th2 response induced by an HIV-1-specific DNA vaccine. J Immunol, 2000. 164(6): p. 3102-11.
30. Simons, J.W., et al., Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer. Cancer Res, 1999. 59(20): p. 5160-8.
31. Ulmer, J.B., et al., Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 1993. 259(5102): p. 1745-9.
32. Wei, C., et al., Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes. Cancer Immunol Immunother, 1996. 42(6): p. 362-8.
33. Correale, P., et al., In vitro generation of human cytotoxic T lymphocytes specific for peptides derived from prostate-specific antigen. J Natl Cancer Inst, 1997. 89(4): p. 293-300.
34. Wei, C., et al., Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy. Proc Natl Acad Sci U S A, 1997. 94(12): p. 6369-74.
35. Overwijk, W.W., et al., Vaccination with a recombinant vaccinia virus encoding a "self" antigen induces autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4(+) T lymphocytes. Proc Natl Acad Sci U S A, 1999. 96(6): p. 2982-7.
36. Johnson, M.A., et al., Isolation and characterization of mouse probasin: An androgen-regulated protein specifically expressed in the differentiated prostate. Prostate, 2000. 43(4): p. 255-62.