(3.238.186.43) 您好!臺灣時間:2021/03/05 21:58
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:廖艾琪
研究生(外文):Liao, Ai-chi
論文名稱:抗血管生成因子之選殖:腫瘤基因療法的應用.
論文名稱(外文):Cloning of the genes of angiostatin and endostatin : Application of the antiangiogenic factors in cancer gene therapy.
指導教授:黃麗華黃麗華引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:107
中文關鍵詞:抗血管生成因子腫瘤基因療法抗血管生成作用重組腺病毒重組反轉錄病毒
外文關鍵詞:Antiangiogenic factorsAngiostatinEndostatinTumor gene therapyAntiangiogenesisRecombinant adenovirusesRecombinant retroviruses
相關次數:
  • 被引用被引用:0
  • 點閱點閱:145
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
基於腫瘤生長及其轉移的發生是必須依賴血管生成的發現,且由於抗血管生成的腫瘤療法具有不易產生抗藥性的特點,利用阻止血管的生成來抑制腫瘤及轉移的發生是近年來積極研究的方向。Angiostatin和endostatin分別是人體中plasminogen和collagen XVIII經蛋白質水解水解後的片段,已有研究指出,這兩個蛋白質片段能有效地抑制內皮細胞增生及腫瘤生長。在腫瘤位置長期維持這些抗血管生成因子的量及濃度是為了讓腫瘤限制在一定的大小之下且處於〝休眠〞的狀態,而欲達到這樣的目的,基因療法無疑是最好的選擇。
基於這樣的理由,我們分別構築了帶有人類angiostatin (HA)、endostatin(HE)和老鼠endostatin(ME) cDNA的重組反轉錄病毒載體。利用這些載體將抗血管生成因子的cDNA轉殖入GP7TB和RT2兩腫瘤細胞株中。首先我們以西方墨點分析法確定這些抗血管生成因子是可以分泌到細胞外表現。接著我們利用共同培養的方法和in vivo腫瘤生長性的實驗測試這些因子的生物活性。在in vitro實驗中雖然只有在會表現HA和HE的腫瘤細胞組中看到抑制內皮細胞增生的現象,但是在in vivo的腫瘤生長性實驗中,我們可以確定這三個抗血管生成因子都是具有生物活性的。
為了以後能直接進行in vivo的治療,我們也構築了帶有HA和ME的重組腺病毒(Ad-HA和Ad-ME)。在以重組腺病毒感染HUVEC細胞來測試這兩個抗血管生成因子的生物活性時,雖然我們只看到Ad-ME能抑制HUVEC細胞增生,並不表示Ad-HA就沒有這樣的效果。因為我們在選殖重組反轉錄病毒和重組腺病毒時所用的cDNA片段來源是一樣,理論上在重組反轉錄病毒中可看到效果,在重組腺病毒中也應該有一樣的效果。
此外,我們也構築了一個特定只在肝癌細胞中表現腺病毒E1蛋白質的質體,並確定此質體確實只在肝癌細胞中表現E1蛋白質以供給重組腺病毒複製所需,而並非是在子宮頸癌細胞中表現。
在本篇論文中,我們製備了帶有抗血管生成因子的重組反轉錄病毒和重組腺病毒,希望能將這些抗血管生成之基因療法的材料和腫瘤的免疫療法搭配使用,以達到治療的加成性。

Based on the observation that solid tumor growth and metastasis are angiogenesis dependent, antiangiogenic tumor therapies have recently attracted intense interest for their broad-spectrum action, low toxicity, and, in the case of direct endothelial target, an absence of drug resistance. To promote tumor regression and to maintain dormancy, anti-angiogenic agents need to be chronically administered. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. Angiostatin and endostatin , the proteolytic fragments of plasminogen and collagen XVIII, respectively, have recently been shown to potently inhibit endothelial proli-feration in vitro and tumor growth in vivo.
As a step toward this goal, we have generated re-combinant retroviral vectors that carry genes coding for human angiostatin(HA), human endo-statin(HE), and mouse endostatin(ME). These retroviral vectors successful-ly transduced angiostatin and endostatin cDNA in two rat tumor cell lines, GP7TB and RT2. We confirmed by Western blotting analysis that the anti-angiogenic factors could be secreted out of the cells. In vitro bioassays indicated that only the tumor cells expressing HA and HE could inhibit HUVE cells proliferation. Nevertheless, all three anti-angiogenic factors exhibited the potentials of regressing tumor in vivo.
We also generated recombinant adenoviral vectors which carried genes coding for HA or ME(Ad-HA or Ad-ME). Using the recombinant adenoviruses to infect HUVE cells at an MOI of 300, the proliferation of HUVE cells were inhibited by Ad-ME, but not by Ad-HA. However, we can't negate the biological activity of Ad-HA currently, because the cDNA fragment was obtained from the same source as that in construction of recombinant retroviral vectors.
We also constructed the pShuttle-AFP-E1 plasmid which could specifically express E1 protein in hepatoma cell lines. We have confirmed that expression of AFP-E1 could limit the replication of adenoviruses in hepatoma cells. Next step we will generate recombinant adenoviruses containing the AFP-E1 fragment. Our purpose is to construct a conditionally replicative adenovirus in order to improve the effects of adenoviral gene therapy.
In conclusion, in this study, we have generated recombinant retroviral and adenoviral vectors carrying antiangiogenic factor cDNAs. These vectors can provide a potential use for tumor gene therapy in combination with immunogene therapy.

【中文摘要】 ---------------------------------------------------------------------------------------------------- i
【英文摘要】 ---------------------------------------------------------------------------------------------------- iii
【縮寫表】 ------------------------------------------------------------------------------------------------------- vi
【導論】 ----------------------------------------------------------------------------------------------------------- 1
壹、血管生成(Angiogenesis)之機制與調控 ------------------------------------------- 1
貳、血管生成和腫瘤生長及轉移(Metastases) ---------------------------------------- 3
參、腫瘤之抗血管生成療法(Antiangiogenesis) --------------------------------------- 6
肆、抗血管生成因子之一---Angiostatin ------------------------------------------------ 7
伍、抗血管生成因子之二---Endostatin ------------------------------------------------- 12
陸、抗血管生成的基因療法 ------------------------------------------------------------- 14
【材料與方法】 ------------------------------------------------------------------------------------------------- 17
材料 --------------------------------------------------------------------------------------------- 17
方法 --------------------------------------------------------------------------------------------- 28
壹、抗血管生成因子(Antiangiogenic factors) cDNA之選殖 --------------- 28
貳、抗血管生成因子在大腸桿菌中的表現 --------------------------------- 39
參、反轉錄病毒的製備 ----------------------------------------------------------- 46
肆、重組腺病毒的製備(recombinant adenoviruses) -------------------------- 56
【結果】 ---------------------------------------------------------------------------------------------------------- 64
壹、抗血管生成因子cDNA之選殖與序列分析 ------------------------------------ 64
貳、在大腸桿菌系統表現、純化抗血管生成因子及其抗體之製備 -------- 65
參、抗血管生成因子穩定表現細胞株之建立及細胞株釋
放之抗血管生成因子的活性測試 ----------------------------------------------- 67
肆、利用重組腺病毒載體表現抗血管生成因子 ---------------------------------- 72
伍、提供特定情況下重組腺病毒複製能力之測試 ------------------------------- 73
【討論】 ---------------------------------------------------- 76
【總結】 ---------------------------------------------------- 83
【圖表】 ---------------------------------------------------- 84
【參考文獻】 ---------------------------------------------- 107

Blezinger, P., Wang, J., Gondo, M., Quezada, A., Mehrens, D., French, M., Singhal, A., Sullivan, S., Rolland, A., Ralston, R., and Min, W. (1999) Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. Nature Biotechnology 17, 343-348.
Boehm, T., Folkman, J., Browder, T., and O'Reilly, M.S. (1997) Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, 404-407.
Brooks, P.C., Silletti, S., Schalscha, T.L.V., Friedlander, M., and Cheresh, D.A. (1998) Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity. Cell 92, 391-400.
Cao, Y., Ji, R.W., Davidson, D., Schaller, J., Marti, D., Sohndel, S., McCance, S.G., O'reilly, M.S., Llinas, M., and Folkman, J. (1996) Kringle domains of human angiostatin. J. Biol. Chem. 271, 29461-29467.
Cao, Y., Chen, A., An, S.S.A., Ji, R.W., Davidson, D., Cao, Y., and Llinas, M. (1997) Kringle 5 of plasminnogen is a novel inhibitor of endothelial cell growth. J. Biol. Chem. 272, 22924-22928.
Cao, Y., O'reilly, M.S., Marshall, B., Flynn, E., Ji, R.W., and Folkman, J. (1998) Expression of angiostatin cDNA in a murine fibrosarcoma suppresses primary tumor growth and produces long-term dormacy of metastases. J. Clin. Invest. 101, 1055-1063.
Chen, B.F., Hwang, L.H., and Chen, D.S. (1993) Characterization of a bicistronic retroviral vector composed of the swine vesicular disease virus internal ribosome entry site. J. Viol. 67, 2142-2148.
Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid quanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159.
Chow, N.H., Hsu, P., Lin, X.Z., Yang, H.B., Chan, S.H., Cheng, K.S., Huang, S.M., and Su, I.J. (1997) Expression of vascular endothelial growth factor in normal liver and hepatocellular carcinoma. Human Pathology 28, 698-703.
Claesson-Welsh, L., Welsh, M., Ito, N., Anand-Apte, B., Soker, S., Zetter, B., O'Reilly, M., and Folkman, J. (1998) Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc. Natl. Acad. Sci. USA 95, 5579-5583.
Clark, W.H., Jr., Elder, D.E., Guerry, D.I., Braitman, L.E., Trock, B.J., Schultz, D., Synnestevdt, M., and Halpern, A.C. (1989) Model predicting survival in stage I melanoma based on tumor progression. J. Natl. Cancer Inst. 81, 1893-1904.
Dhanabal, M., Ramchandran, R., Volk, R., Stillman, I.E., Lombardo, M., Iruela-Arispe, M.L., Simons, S., and Sukhatme, V.P. (1999) Endostatin:Yeast production, mutants, and anti-tumor effect in renal cell carcinoma. Cancer Res. 59, 189-197.
Dong, Z., Jumar, R., Yang, X., and Fidler, I.J. (1997) Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell 88, 801-810.
Fidler I.J. (1991) Cancer metastasis. British Medical Bulletin 47, 157-177.
Fidler, I.J., and Ellis, L.M. (1994) The implication of angiogenesis for the biology and therapy of cancer metastasis. Cell 79, 185-188.
Folkman J. (1971) Tumor angiogenesis:therapeutic implications. New Engl. J. Med. 285, 1182-1186.
Folkman, J. (1986) How is blood vessel growth regulated in normal and neoplastic tissue? Cancer Res. 46, 467-473.
Folkman, J., and Klagsbrun, M. (1987) Angiogenic factors. Science 235, 442-447.
Folkman, J. (1990) What is the evidence that tumors are angio-genesis dependent? J. Natl. Cancer Inst. 82, 4-6.
Folkman, J., and Shing, Y. (1992) Angiogenesis. J. Biol. Chem. 267, 10931-10934.
Folkman, J. (1996a) Fighting cancer by attacking its blood supply. Scientific American 275, 150-154.
Folkman, J. (1996b) Tumor angiogenesis and tissue factor. Nature Medicine 2, 167-168.
Fong T.A.T., Shawver, L.K., Sun, L., Tang, C., App, H., Powell, T.J., Kim, Y.H., Schreck, R., Wang, X., Risau, W., Ullrich, A., Hirth, K.P., and McMahon, G. (1999) SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor(Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res. 59, 99-106.
Forsgren, M., Raden, B., Israelsson, M., Laesson, K., and Heden, L.O. (1987) Molecular cloning and characterization of a full-length cDNA for human plasminogen. FEBS Lett. 213, 254-260.
Gately, S., Twardowski, P., Stack, M.S., Patrick, M., Boggio, L., Cundiff, D.L., Schnaper, H.W., Madison, L., Volpert, O., Bouck, N., Enghild, J., Kwaan, H.C., and Soff, G.A. (1996) Human prostata carcinoma cells express enzymatic activity that converts human plasminogen to angiogenesis inhibitor, angiostatin. Cancer Research 58, 4887-4890.
Gately, S., Twardowski, P., Stack, M.S., Cundiff, D.L., Grella, D., Castellino, F.J., Enghild, J., Keaan, H.C., Lee, F., Kramer, R.A., Volpert, O., Bouck, N., and Soff, G.A. (1997) The mechanism of cancer-mediated conversion of plasminogen to the angiogensis inhibitor angiostatin. Proc. Natl. Acad. Sci. USA 94, 10868-10872.
Graham, F.L., and Smiley, J. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59-72.
Griscelli, F., Li, H., Bennaceur-Griscelli, A., Soria, J., Opolon, P., Soria, C., Perricaudet, M., Yeh, P., and Lu, H. (1998) Angiostatin gene transfer:inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest. Proc. Natl. Acad. Sci. USA 95, 6367-6372.
Hanahan, D., and Folkman, J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-364.
Hanahan, D. (1997) Signaling vascular morphogenesis and maintenance. Science 277, 48-50.
Hanahan, D. (1998) A flanking attack on cancer. Nature Medicine 4, 13-14.
He, T.-C., Zhou, S., Costa, L.T.D., Yu, J., Kinziler, K.W., and Vogelstein, B. (1998) A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 95, 2509-2514.
Hobson, B., and Denekamp, J. (1984) Endothelial proliferation in tumor and normal tissues:continuous labelling studies. Br. J. Cancer 49, 405-413.
Holmgren, L., O'Reilly, M.S., and Folkman, J. (1995) Dormancy of micrometastases:balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nature Med. 1, 149-153.
Hori, A., Sasada, R., Matsutani, E., Naito, K., Sakura, Y., Fujita, T., and Kozai, Y. (1991) Suppression of solid tumor growth by immononeutralizing monoclonal antibody against human basic fibroblast growth factor. Cancer Res. 51, 6180-6184.
Hsieh, C.L., Chen, B.F., Wang, C.C., Liu, H.H., Chen, D.S., and Hwang, L.H. (1995) Improved gene expression by a modified bicistronic retroviral vector. Biochemical and Biophysical Research Communications 214, 910-917.
Kim, K.J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H.S. et al. (1993) Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362, 841-844.
Klagsbrun, M. (1999) Angiogenesis and cancer. Cancer Res. 59, 487-490.
Kong, H.L., Hecht, D., Song, W., Kovesdi, I., Hackett, N.R., Yayon, A., and Crystal, R.G. (1998) Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor. Human Gene Therapy 9, 823-833.
Maisonpierre, P.C., Suri, C., Jones, P.F., Bartunkova, S., Wiegand, S.J., Radziejewski, C., Compton, D., McClain, J., Aldrich, T.H., Papadopoulos, N., Daly, T.J., Davis, S., Sato, T.N., and Yancopoulos, G.D. (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55-60.
Markowitz, D., Goff, S., and Bank, A. (1988) A safe packaging line for gene transfer:separating viral genes on the different plasmids. J. Virol. 62, 1120-1124.
Mauceri, H.J., Hanna, N.N., Beckett, M.A., Gorski, D.H., Staba, M.J., Stellato, K.A., Bigelow, K., Heimann, R., Gately, S., Dhanabal, M., Soff, G.A., Sukhatme, V.P., Kufe, D.W., and Weichselbaum, R.R. (1998) Combined effects of angiostatin and ionizing radiation in antitumour therapy. Nature 394, 287-290.
Mise, M., Arii, S., Higashituji, H., Furutani, M., Niwano, M., Harada, T., Ishigami, S., Toda, Y., Nakayama, H., Fukumoto, M., Fujita, J., and Imamura, M. (1996) Clinical significance of vascular endothelial growth factor and basic fibroblast growth factor gene expression in liver tumor. Hepatology 23, 455-464.
Moser, T.L., Stack, M.S., Asplin, I., Enghild, J.J., Hojrup, P., Everitt, L., Hubchak, S., Schnaper, H.W., and Pizzo, S.V. (1999) Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc. Natl. Acad. Sci. USA 96, 2811-2816.
Nguyen, J.T., Wu, P., Clouse, M.E., Hlatky, L., and Terwilliger, E.F.(1998) Adeno-associated virus-mediated delivery of antiangiogenic factors as an antitumor strategy. Cancer research 58, 5673-5677.
Obeso, J., Weber, J., and Auerbach, R. (1990) A hemangio-endothelioma-derived cell line:its use as a model for study of endothelial cell biology. Lab.Invest. 63, 259-269.
Oh, S.P., Warman, M.L., Seldin, M.F., Cheng, S.D., Knoll, J.H.M.,Timmons, S., Olsen, B.R. (1994) Cloning of cDNA and genomic DNA encoding human type XVIII collagen and locali-zation of the a1(XVIII) collagen gene to mouse chromosome 10 and human chromosome 21. Genomics 19, 494-499.
O'reilly, M.S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R.A., Moses, M., Lane, W.S., Cao, Y., Sage, E.H., and Folkman, J. (1994) Angiostatin : a novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma. Cell 79, 315-328.
O'reilly, M., Rosenthal, R., Sage, E.H., Smith, S., Holmgren, L., Moses, M., Shing, Y., and Folkman, J. (1993) The suppression of tumor metastases by a primary tumar. Surg. Forum. 44, 474-476.
O'reilly, M.S., Holmgren, L., Chen, C., and Folkman, J. (1996) Angiostatin induces and sustains dormancy of human primary tumors in mice. Nature Medicine 2, 689-692.
O'Reilly, M.S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W.S., Flynn, E., Birkhead, J.R., Olsen, B.R., and Folkman, J. (1997) Endostatin:an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285.
Pluda, J.M. (1997) Tumor-associated angiogenesis:mechanism, clinical implication, and therapeutic strategies. Seminars in Oncology 24, 203-218.
Pluda, J.M., and Parkinson, D.R. (1996) Clinical implications of tumor-associated neovascularization and current antiangio-genic strategies for the treatment of malignancies of pancreas. Cancer Supplement 78, 680-687.
Poste, G., and Fidler, I.J. (1980) The pathogenesis of cancer metastasis. Nature 283, 139-145.
Rak, J., Filmus, J., Finkenzeller, G., Grugel, S., Marme, D., and Kerbel, R.S. (1995) Oncogene as inducer of tumor angiogenesis. Cancer and Metastasis Reviews 14, 263-277.
Relf, M., LeJeune, S., Scott, P.A.E., Fox, S., Smith, K., Leek, R., Moghaddam, A., Whitehouse, R., Bicknell, R., and Harris, A.L. (1997) Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor b-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis. Cancer Res. 57, 963-969.
Risau, W. (1997) Mechanisms of angiogenesis. Nature 386, 671-674.
Shalaby, F. et al. (1995) Failure of blood-island formation and vasculogenesis in flk-1-deficient mice. Nature 376, 62-66.
Sim, B.K.L., O'Reilly, M.S., Liang, H., Fortier, A.H., He, W., Madsen, J.W., Lapcevich, R., and Nacy, C.A. (1997) A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer. Cancer Res. 57, 1329-1334.
Stathakis, P., Fitzgerald, M., Matthias, L.J., Chesterman, C.N., and Hogg, P.J. (1997) Generation of angiostatin by reduction and proteolysis of plasmin. J. Biol. Chem. 272, 20641-20645.
Tanaka, T., Manome, Y., Wen, P., Kufe, D.W., Fine, H.A. (1997) Viral vector-mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth. Nature Med. 3, 437-442.
Tanaka, T., Cao, Y., Folkman, J., and Fine, H.A. (1998) Viral vector-targeted antiangiogenic gene therapy utilizing an angiostatin complementary DNA. Cancer Res. 58, 3362-3369.
Voest, E.E., Kenyon, B.M., O'Reilly, M.S., Truitt, G., D'Amato, R.J., and Folkman, J. (1995) Inhibition of angiogenesis in vivo by interleukin 12. J. Natl. Cancer Inst. 87, 581-586.
Wu, Z., O'Reilly, M.S., Folkman, J., and Shing, Y. (1997) Suppression of tumor growth with recombinant murine angiostatin. Biochem. Biophys. Res. Commun. 236, 651-654.
Yancopoulos, G., Klagsbrun, M., and Folkman, J. (1998) Vasculogenesis, angiogenesis, and growth factors:ephrins enter the fray at the border. Cell 93, 661-664.
Yeh, C.H., Peng, H.C., and Huang, T.F. (1998) Accutin, a new disintegrin, inhibits angiogenesis in vitro and in vivo by acting as integrin aVb3 antagonist and inducing apoptosis. Blood 92, 3268-3276.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊
 
系統版面圖檔 系統版面圖檔