系統識別號 U0026-0812200915170663
論文名稱(中文) 使用吲哚胺2,3雙加氧酶小髮夾型核醣核酸治療原位肝癌
論文名稱(英文) Treatment of Indoleamine 2,3-dioxygenase shRNA Has Therapeutic Efficacy in an Orthotopic Hepatoma Model
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 97
學期 2
出版年 98
研究生(中文) 黃秭庭
研究生(英文) Tzu-Ting Huang
電子信箱 s1696407@mail.ncku.edu.tw
學號 s1696407
學位類別 碩士
語文別 英文
論文頁數 47頁
口試委員 口試委員-吳昭良
中文關鍵字 原位肝癌  3雙加氧酶  吲哚胺2  基因槍 
英文關鍵字 gene gun  Orthotopic hepatoma model  Indoleamine 2  3-dioxygenase 
中文摘要 肝癌是世界上最常發生的惡性腫瘤之一,而且其預後相當差。目前有相當多的證據指出,癌症特異的免疫耐受性對癌症的進程具有相當重要的影響力。吲哚胺2,3雙加氧酶(indoleamine 2,3-dioxygenase, IDO)是代謝色胺酸的速限酵素,不僅對腫瘤的進程有重要影響,也能使腫瘤逃脫免疫攻擊,而在腫瘤附近淋巴結可以觀察到過量吲哚胺2,3雙加氧酶的表現。因此,若抑制吲哚胺2,3雙加氧酶的表現則可能抑制肝癌的進程進而獲得療效。實驗室之前的研究指出,吲哚胺2,3雙加氧酶小髮夾型核醣核酸在許多動物模式下可以有效抑制腫瘤的生成,然而,皮下注射所生成的腫瘤與原位癌在整體免疫中有所不同。我們利用手術的方式直接將肝癌細胞ML14a打入小鼠的肝葉以建立一個原位肝癌的動物模式,並可在第七天時看見腫瘤的形成,同時也發現了與假手術組別小鼠的腸繫膜淋巴結相比較,的確有大量吲哚胺2,3雙加氧酶表現。此時便利用基因槍的方式遞送吲哚胺2,3雙加氧酶小髮夾型核醣核酸,經由基因槍治療之後將小鼠犧牲並秤肝臟的重量。從結果發現施予吲哚胺2,3雙加氧酶小髮夾型核醣核酸組別的小鼠獲得顯著的治療效果,為了進一步探討可能的機制,利用免疫染色的方式觀察,發現在施予吲哚胺2,3雙加氧酶小髮夾型核醣核酸的組別有較多的淋巴細胞浸潤以及較高的細胞毒殺效果,暗示著施予吲哚胺2,3雙加氧酶小髮夾型核醣核酸可以誘發免疫反應。另外,施予吲哚胺2,3雙加氧酶小髮夾型核醣核酸也可以有效抑制癌細胞在肺部的轉移。未來或許可以藉著與吲哚胺2,3雙加氧酶的抑制劑Dextro-1-Methyl Tryptophan (D-1MT),進行共同治療以增強其療效。
英文摘要 Hepatocellular carcinoma (HCC, also called hepatoma) is one of the most prevalent malignancies in the world, and the prognosis of hepatoma is poor. There is accumulating evidence showing that tumor specific tolerance influences tumor progression. Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme of tryptophan metabolism, is important in tumor progression and can contribute to tumor immune escape. Over-expression of IDO can be observed in the tumor-draining lymph node. This findings imply that blockage of IDO may have the therapeutic efficacy on the progression of hepatoma. Our previous results have demonstrated that IDO shRNA has therapeutic effect on inhibiting the growth of tumors implanted subcutaneously in several animal tumor models. However, the immunological environment is quite different when tumor is implanted subcutaneously. We established an orthotropic hepatoma animal model by directly inoculating ML14a cells into the liver. The tumor was formed at day 7 after intra-hepatic injection. We found that expression of IDO was elicited compared with the sham group in the mesenteric lymph node. We then treated the mice with IDO shRNA via gene gun and sacrificed them at the defined time intervals to measure the size of liver tumor. Treatment of IDO shRNA exerted therapeutic efficacy in the orthotropic hepatoma model. To further investigate the possible mechanism, we performed Immunohistochemical staining and found that there were more tumor-infiltrating lymphocytes and higher cytotoxic lysis percentage in the IDO shRNA group, suggesting that treatment of IDOshRNA may trigger an immunological response. Furthermore, we observed that IDO shRNA vaccination also has therapeutic efficacy in a metastatic tumor model. In the future, combination of the IDO inhibitor, Dextro-1-Methyl Tryptophan (D-1MT), with IDOshRNA may be further explored to enhance the therapeutic effect in the orthotropic hepatoma model.
論文目次 Abstract
Page number list _________________________________________ Ⅰ
Introduction_____________________________________________ 1
Materials and methods____________________________________ 8
(A) Materials__________________________________________ 8
(a) Cell culture_____________________________________ 8
(b) Preparation of plasmid DNA_______________________ 10
(c) Immunohistochemistry___________________________ 11
(B) Methods __________________________________________ 13
(a) Animals________________________________________ 13
(b) Cell lines and animal models________________________ 13
(c) Intracellular cytokine staining_______________________ 14
(d) Preparation of DNA vaccine ________________________ 15
(e) Treatment protocols_______________________________ 15
(f) In vivo bioluminescence imaging ____________________ 16
(g) Immunohistochemical staining for infiltrated lymphocytes _ .16
(h) In vitro cytotoxicity assay of lymphocytes_____________. 16
(i) Statistics _______________________________________. 17
Results_________________________________________________. 18
(a) Therapeutic efficacy of IDO shRNA in subcutaneous hepatoma model_____________________________________________ 18
(b) Therapeutic efficacy of IDO shRNA in orthotopic hepatoma model __________________________________________________ 18
(c) Therapeutic efficacy of IDO shRNA in metastatic animal model_____________________________________________ 20
Discussion______________________________________________ 22
References______________________________________________ 27
Table __________________________________________________ 34
Figures ________________________________________________ 36
Appendix ______________________________________________ 46
參考文獻 1. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557-2576.
2. Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P, Calvisi DF, et al. A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat Med 2006;12:410-416.
3. Kurokawa Y, Matoba R, Takemasa I, Nagano H, Dono K, Nakamori S, et al. Molecular-based prediction of early recurrence in hepatocellular carcinoma. J Hepatol 2004;41:284-291.
4. Giannelli G, Bergamini C, Marinosci F, Fransvea E, Quaranta M, Lupo L, et al. Clinical role of MMP-2/TIMP-2 imbalance in hepatocellular carcinoma. Int J Cancer 2002;97:425-431.
5. Fransvea E, Angelotti U, Antonaci S, Giannelli G. Blocking transforming growth factor-beta up-regulates E-cadherin and reduces migration and invasion of hepatocellular carcinoma cells. Hepatology 2008;47:1557-1566.
6. Siegel PM, Massague J. Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer 2003;3:807-821.
7. Befeler AS, Di Bisceglie AM. Hepatocellular carcinoma: diagnosis and treatment. Gastroenterology 2002;122:1609-1619.
8. Sun K, Wang L, Zhang Y. Dendritic cell as therapeutic vaccines against tumors and its role in therapy for hepatocellular carcinoma. Cell Mol Immunol 2006;3:197-203.
9. Wilhelm S, Carter C, Lynch M, Lowinger T, Dumas J, Smith RA, et al. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 2006;5:835-844.
10. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378-390.
11. Sharma MD, Baban B, Chandler P, Hou DY, Singh N, Yagita H, et al. Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase. J Clin Invest 2007;117:2570-2582.
12. Igney FH, Krammer PH. Immune escape of tumors: apoptosis resistance and tumor counterattack. J Leukoc Biol 2002;71:907-920.
13. Chen G, Luo DZ, Liu L, Feng ZB, Guo F, Li P. Hepatic local micro-environmental immune status in hepatocellular carcinoma and cirrhotic tissues. West Indian Med J 2006;55:403-408.
14. Unitt E, Rushbrook SM, Marshall A, Davies S, Gibbs P, Morris LS, et al. Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells. Hepatology 2005;41:722-730.
15. Dougan M, Dranoff G. Immune therapy for cancer. Annu Rev Immunol 2009;27:83-117.
16. Peachman KK, Rao M, Alving CR. Immunization with DNA through the skin. Methods 2003;31:232-242.
17. Becker FF, Klein KM, Wolman SR, Asofsky R, Sell S. Characterization of primary hepatocellular carcinomas and initial transplant generations. Cancer Res 1973;33:3330-3338.
18. Meng WS, Butterfield LH, Ribas A, Dissette VB, Heller JB, Miranda GA, et al. alpha-Fetoprotein-specific tumor immunity induced by plasmid prime-adenovirus boost genetic vaccination. Cancer Res 2001;61:8782-8786.
19. Tu CF, Lin CC, Chen MC, Ko TM, Lin CM, Wang YC, et al. Autologous neu DNA vaccine can be as effective as xenogenic neu DNA vaccine by altering administration route. Vaccine 2007;25:719-728.
20. Lin CC, Yen MC, Lin CM, Huang SS, Yang HJ, Chow NH, et al. Delivery of noncarrier naked DNA vaccine into the skin by supersonic flow induces a polarized T helper type 1 immune response to cancer. J Gene Med 2008;10:679-689.
21. Cunha GR, Matrisian LM. It's not my fault, blame it on my microenvironment. Differentiation 2002;70:469-472.
22. Laconi E, Doratiotto S, Vineis P. The microenvironments of multistage carcinogenesis. Semin Cancer Biol 2008;18:322-329.
23. Ingber DE. Can cancer be reversed by engineering the tumor microenvironment? Semin Cancer Biol 2008;18:356-364.
24. Schrocksnadel H, Baier-Bitterlich G, Dapunt O, Wachter H, Fuchs D. Decreased plasma tryptophan in pregnancy. Obstet Gynecol 1996;88:47-50.
25. Hirata F, Ohnishi T, Hayaishi O. Indoleamine 2,3-dioxygenase. Characterization and properties of enzyme. O2- complex. J Biol Chem 1977;252:4637-4642.
26. Mellor AL, Munn DH. Tryptophan catabolism and T-cell tolerance: immunosuppression by starvation? Immunol Today 1999;20:469-473.
27. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998;281:1191-1193.
28. Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, Falorni A, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol 2002;3:1097-1101.
29. Mellor AL, Munn DH. Tryptophan catabolism and regulation of adaptive immunity. J Immunol 2003;170:5809-5813.
30. Munn DH, Sharma MD, Hou D, Baban B, Lee JR, Antonia SJ, et al. Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J Clin Invest 2004;114:280-290.
31. Prendergast GC. Immune escape as a fundamental trait of cancer: focus on IDO. Oncogene 2008;27:3889-3900.
32. Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA. Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J Immunol 2000;164:3596-3599.
33. Munn DH, Sharma MD, Lee JR, Jhaver KG, Johnson TS, Keskin DB, et al. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 2002;297:1867-1870.
34. Liu Z, Dai H, Wan N, Wang T, Bertera S, Trucco M, et al. Suppression of memory CD8 T cell generation and function by tryptophan catabolism. J Immunol 2007;178:4260-4266.
35. Chen W, Liang X, Peterson AJ, Munn DH, Blazar BR. The indoleamine 2,3-dioxygenase pathway is essential for human plasmacytoid dendritic cell-induced adaptive T regulatory cell generation. J Immunol 2008;181:5396-5404.
36. Munn DH, Mellor AL. Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J Clin Invest 2007;117:1147-1154.
37. Yen MC, Lin CC, Chen YL, Huang SS, Yang HJ, Chang CP, et al. A novel cancer therapy by skin delivery of indoleamine 2,3-dioxygenase siRNA. Clin Cancer Res 2009;15:641-649.
38. Chang CJ, Tai KF, Roffler S, Hwang LH. The immunization site of cytokine-secreting tumor cell vaccines influences the trafficking of tumor-specific T lymphocytes and antitumor efficacy against regional tumors. J Immunol 2004;173:6025-6032.
39. Killion JJ, Radinsky R, Fidler IJ. Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer Metastasis Rev 1998;17:279-284.
40. Crispe IN. Hepatic T cells and liver tolerance. Nat Rev Immunol 2003;3:51-62.
41. Huang L, Soldevila G, Leeker M, Flavell R, Crispe IN. The liver eliminates T cells undergoing antigen-triggered apoptosis in vivo. Immunity 1994;1:741-749.
42. Salmi M, Jalkanen S. How do lymphocytes know where to go: current concepts and enigmas of lymphocyte homing. Adv Immunol 1997;64:139-218.
43. Gravier R, Dory D, Laurentie M, Bougeard S, Cariolet R, Jestin A. In vivo tissue distribution and kinetics of a pseudorabies virus plasmid DNA vaccine after intramuscular injection in swine. Vaccine 2007;25:6930-6938.
44. Stoecklinger A, Grieshuber I, Scheiblhofer S, Weiss R, Ritter U, Kissenpfennig A, et al. Epidermal langerhans cells are dispensable for humoral and cell-mediated immunity elicited by gene gun immunization. J Immunol 2007;179:886-893.
45. Terme M, Chaput N, Combadiere B, Ma A, Ohteki T, Zitvogel L. Regulatory T cells control dendritic cell/NK cell cross-talk in lymph nodes at the steady state by inhibiting CD4+ self-reactive T cells. J Immunol 2008;180:4679-4686.
46. Torgersen J, Collas P, Alestrom P. Gene-Gun-Mediated Transfer of Reporter Genes to Somatic Zebrafish (Danio rerio) Tissues. Mar Biotechnol (NY) 2000;2:293-300.
47. Brown RR, Ozaki Y, Datta SP, Borden EC, Sondel PM, Malone DG. Implications of interferon-induced tryptophan catabolism in cancer, auto-immune diseases and AIDS. Adv Exp Med Biol 1991;294:425-435.
48. Baban B, Chandler P, McCool D, Marshall B, Munn DH, Mellor AL. Indoleamine 2,3-dioxygenase expression is restricted to fetal trophoblast giant cells during murine gestation and is maternal genome specific. J Reprod Immunol 2004;61:67-77.
49. Uyttenhove C, Pilotte L, Theate I, Stroobant V, Colau D, Parmentier N, et al. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 2003;9:1269-1274.
50. Salter M, Pogson CI. The role of haem in the regulation of rat liver tryptophan metabolism. Biochem J 1986;240:259-263.
51. Ball HJ, Sanchez-Perez A, Weiser S, Austin CJ, Astelbauer F, Miu J, et al. Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice. Gene 2007;396:203-213.
52. Metz R, Duhadaway JB, Kamasani U, Laury-Kleintop L, Muller AJ, Prendergast GC. Novel tryptophan catabolic enzyme IDO2 is the preferred biochemical target of the antitumor indoleamine 2,3-dioxygenase inhibitory compound D-1-methyl-tryptophan. Cancer Res 2007;67:7082-7087.
53. Hou DY, Muller AJ, Sharma MD, DuHadaway J, Banerjee T, Johnson M, et al. Inhibition of indoleamine 2,3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with antitumor responses. Cancer Res 2007;67:792-801.
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