臺灣博碩士論文加值系統

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

陸、參考文獻
Allison, J., Georgiou, H.M., Strasser, A., and Vaux, D.L. (1997). Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts. Proc Natl Acad Sci U S A 94, 3943-7.
Bajorath, J. (1999). Identification of the ligand binding site in Fas (CD95) and analysis of Fas-ligand interactions. Proteins 35, 475-482.
Bellgrau, D., Gold,D., Selawry, H., Moore, J., Franzusoff, A., and Duke, R.C. (1995). A role for CD95 ligand in preventing graft rejection. Nature 377, 630-2.
Brunner, T., Kasibhatla, S., Pinkoski, M.J., Frutschi,C., Yoo, N.J., Echeverri,F., Mahboubi, A., and Green, D.R. (2000). Expression of Fas ligand in activated T cells is regulated by c-Myc. J Biol Chem 275, 9767-72.
Chan, H., Bartos, D.P., and Owen-Schaub, L.B. (1999). Activation-dependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50-p65 recruitment. Mol Cell Biol 19, 2098-108.
Chen, J.J., Sun, Y., and Nabel, G.J. (1998). Regulation of the proinflammatory effects of Fas ligand (CD95L). Science 282, 1714-7.
Chinnaiyan, A.M., O'Rourke, K., Tewari, M., and Dixit, V.M. (1995). FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81, 505-12.
Chow, W.A., Fang, J.J., and Yee, J.K. (2000). The IFN regulatory factor family participates in regulation of Fas ligand gene expression in T cells. J Immunol 164, 3512-8.
Deveraux, Q.L., Roy,N., Stennicke, H.R., Van Arsdale,T., Zhou,Q., Srinivasula, S.M., Alnemri, E.S., Salvesen, G.S., and Reed, J.C. (1998). IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. Embo J 17, 2215-23.
Dinarello, C.A. (1998). Interleukin-1 beta, interleukin-18, and the interleukin-1 beta converting enzyme. Ann N Y Acad Sci 856, 1-11.
Drozdzik, M., Qian, C., Lasarte, J.J., Bilbao, R., and Prieto, J. (1998). Antitumor effect of allogenic fibroblasts engineered to express Fas ligand (FasL). Gene Ther 5, 1622-30.
Fisher, G.H., Rosenberg, F.J., Straus, S.E., Dale, J.K., Middleton, L.A., Lin, A.Y., Strober, W., Lenardo, M.J., and Puck, J.M. (1995). Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81, 935-46.
Griffith, T.S., Brunner, T., Fletcher, S.M., Green, D.R., and Ferguson, T.A. (1995). Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 270, 1189-92.
Hohlbaum, A.M., Moe, S., and Marshak-Rothstein, A. (2000). Opposing effects of transmembrane and soluble Fas ligand expression on inflammation and tumor cell survival. J Exp Med 191, 1209-20.
Hsu, S.C., Gavrilin, M.A., Lee, H.H., Wu, C.C., Han, S.H., and Lai, M.Z. (1999). NF-kappa B-dependent Fas ligand expression. Eur J Immunol 29, 2948-56.
Huang, D.C., Cory, S., and Strasser, A. (1997). Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. Oncogene 14, 405-14.
Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J.L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L.E., and Tschopp, J. (1997). Inhibition of death receptor signals by cellular FLIP. Nature 388, 190-5.
Itoh, N., Tsujimoto, Y., and Nagata, S. (1993). Effect of bcl-2 on Fas antigen-mediated cell death. J Immunol 151, 621-7.
Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, M., Seto, Y., and Nagata, S. (1991). The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66, 233-243.
Kang, S.M., Schneider, D.B., Lin, Z., Hanahan, D., Dichek, D.A., Stock, P.G., and Baekkeskov, S. (1997). Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction. Nat Med 3, 738-43.
Keane, M.M., Ettenberg, S.A., Lowrey, G.A., Russell, E.K., and Lipkowitz, S. (1996). Fas expression and function in normal and malignant breast cell lines. Cancer Res 56, 4791-8.
Korbutt, G.S., Elliott, J.F., and Rajotte, R.V. (1997). Cotransplantation of allogeneic islets with allogeneic testicular cell aggregates allows long-term graft survival without systemic immunosuppression. Diabetes 46, 317-22.
Kuida, K., Lippke, J.A., Ku, G., Harding, M.W., Livingston, D.J., Su, M.S., and Flavell, R.A. (1995). Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 267, 2000-3.
Lau, H.T., Yu,M., Fontana, A., and Stoeckert, C.J., Jr. (1996). Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice. Science 273, 109-12.
Lepple-Wienhues, A., Belka, C., Laun, T., Jekle, A., Walter, B., Wieland, U., Welz, M., Heil, L., Kun, J., Busch, G., Weller, M., Bamberg, M., Gulbins, E., and Lang, F. (1999). Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids. Proc Natl Acad Sci U S A 96, 13795-800.
Li,X.R., Chong,A.S., Wu,J., Roebuck,K.A., Kumar,A., Parrillo, J.E., Rapp,U.R., Kimberly, R.P., Williams, J.W., and Xu, X. (1999). Transcriptional regulation of Fas gene expression by GA-binding protein and AP-1 in T cell antigen receptor.CD3 complex-stimulated T cells. J Biol Chem 274, 35203-10.
Lynch, D.H., Ramsdell, F., and Alderson, M.R. (1995). Fas and FasL in the homeostatic regulation of immune responses. Immunol Today 16, 569-74.
Medema, J.P., Scaffidi, C., Kischkel, F.C., Shevchenko, A., Mann, M., Krammer, P.H., and Peter, M.E. (1997). FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). Embo J 16, 2794-804.
Miwa,K., Asano, M., Horai, R., Iwakura, Y., Nagata, S., and Suda, T. (1998). Caspase 1-independent IL-1beta release and inflammation induced by the apoptosis inducer Fas ligand. Nat Med 4, 1287-92.
Mountz, J.D., Zhou, T., Bluethmann, H., Wu, J., and Edwards, C.K.3. (1994). Apoptosis defects analyzed in TcR transgenic and fas transgenic lpr mice. Int Rev Immunol 11, 321-42.
Nagata, S. (1997). Apoptosis by death factor. Cell 88, 355-365.
Nagata, S. and Golstein, P. (1995). The Fas death factor. Science 267, 1449-1456.
O'Connell, J., Bennett, M.W., Nally, K., O'Sullivan, G.C., Collins, J.K., and Shanahan, F. (2000). Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis. J Cell Physiol 185, 331-8.
O'Connell, J., Houston, A., Bennett, M.W., O'Sullivan, G.C., and Shanahan, F. (2001). Immune privilege or inflammation? Insights into the Fas ligand enigma. Nat Med 7, 271-4.
Oehm, A., Behrmann, I., Falk,W., Pawlita, M., Maier, G.K.C., Li-Weber, M., Richards, S., Dhein, J., Trauth, B.C., Ponstingl, H., and Krammer, P.H. (1992). Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily: sequence identify with the Fas antigen. J Biol Chem 267, 10709-10715.
Orlinick, J.R., Elkon, K.B., and Chao, M. (1997). Separate domain of human Fas ligand dictate self-association and receptor binding. J Biol Chem 272, 32222-32229.
Peter, M.E., Kischkel, F.C., Scheuerpflug, C.G., Medema, J.P., Debatin,K.M., and Krammer, P.H. (1997). Resistance of cultured peripheral T cells towards activation-induced cell death involves a lack of recruitment of FLICE (MACH/caspase 8) to the CD95 death-inducing signaling complex. Eur J Immunol 27, 1207-12.
Rieux-Laucat, F., Le Deist,F., Hivroz,C., Roberts, I.A., Debatin, K.M., Fischer, A., and de Villartay, J.P. (1995). Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268, 1347-9.
Ruemmele, F.M., Russo, P., Beaulieu, J., Dionne, S., Levy, E., Lentze, M.J., and Seidman, E.G. (1999). Susceptibility to FAS-induced apoptosis in human nontumoral enterocytes: role of costimulatory factors. J Cell Physiol 181, 45-54.
Schnedier, P., Bodmer, J.-L., Holler, N., Mattmann, C., Scuderi,P ., Terskikh, A., Peitsch, M.C., and Tschopp, J. (1997). Characterization of Fas(Apo-1, CD95)-Fas ligand interaction. J Biol Chem 272, 18827-18833.
Seino, K., Kayagaki, N., Okumura, K., and Yagita, H. (1997). Antitumor effect of locally produced CD95 ligand. Nat Med 3, 165-70.
Starling, G.C., J., B., Emswiler, J., Ledbetter, J.A., Aruffo, A., and Kiener, P.A. (1997). Identification of amino acid residues important for ligand binding to Fas. J Exp Med 185, 1487-1492.
Stohl, W., Xu, D., Starling, G.C., Casali, P., and Kiener, P.A. (2000). Promotion of activated human B cell apoptosis and inhibition of Ig production by soluble CD95 ligand: CD95-based downregulation of Ig production need not culminate in activated B cell death. Cell Immunol 203, 1-11.
Strand, S., Hofmann, W.J., Hug, H.M.M., Otto, G., Strand, D., Mariani, S.M., Stremmel, W., Krammer, P.H., and Galle, P.R. (1996). Lymphocyte apoptosis induced by CD95（APO-1/Fas）ligand expressing tumor cells-A mechanism of immune evasion. Nature Med 2, 1361-1366.
Suda, T., Takahashi, T., Golstein, P., and and Nagata, S. (1993). Molecular cloning and expression of the Fas ligand：a novel member of the tumor necrosis factor family. Cell 75, 1169-1178.
Sumida, T., Matsumoto, I., Murata, H., Namekawa, T., Matsumura, R., Tomioka, H., Iwamoto, I., Saito, Y., Mizushima, Y., Hasunuma, T., Maeda, T., and Nishioka, K. (1997). TCR in Fas-sensitive T cells from labial salivary glands of patients with Sjogren's syndrome. J Immunol 158, 1020-5.
Tanaka, M., Itai, T., Adachi, M., and Nagata, S. (1998). Downregulation of Fas ligand by shedding. Nat Med 4, 31-6.
Yanagisawa, J., Takahashi, M., Kanki, H., Yano-Yanagisawa, H., Tazunoki, T., Sawa, E., Nishitoba, T., Kamishohara, M., Kobayashi, E., Kataoka, S., and Sato, T. (1997). The molecular interaction of Fas and FAP-1. A tripeptide blocker of human Fas interaction with FAP-1 promotes Fas-induced apoptosis. J Biol Chem 272, 8539-45.