臺灣博碩士論文加值系統

高壓氧氣治療 (hyperbaric oxygen therapy, HBOT) 是指在大於1絕對大氣壓 (atmosphere absolute, ATA) 的治療艙內給予患者間歇性吸入100%的氧氣。臨床上常用來治療一氧化碳中毒、問題傷口、減壓症等。由於高壓氧氣治療對腫瘤細胞的影響並不明確，加上對於高濃度的氧氣有促進細胞生長作用的疑慮，使得高壓氧氣治療一直被列為癌症治療的相對禁忌症。而目前的研究報告也指出腫瘤細胞在快速生長的過程中，壞死區域周圍會產生缺氧的情形，另外，缺氧也會造成基因不穩定及突變，進一步增加腫瘤細胞抵抗走向細胞凋亡的能力。除此之外，缺氧亦會誘發缺氧誘發因子 (Hypoxia-Inducible Factor-1, HIF-1)進而造成VEGF (Vascular Endothelial Growth Factor)、Met (tyrosine kinase receptor) 等因子過度表現，使得腫瘤細胞持續增生甚至轉移。本實驗的目的在建立腫瘤缺氧動物模式，探討給予高壓氧氣治療是否可以改善腫瘤缺氧表現進而抑制腫瘤生長甚至轉移。
實驗分為兩部份進行，第一部分首先觀察腫瘤細胞在生長過程中體積變化與缺氧表現，並確立腫瘤缺氧模式，利用異種移植腫瘤細胞至SCID mice，待腫瘤細胞生長至第30、45天時，分別犧牲老鼠，利用免疫組織化學染色法觀察腫瘤細胞內部缺氧表現與血管分佈情形，同時觀察腫瘤體積變化。第二部份則待腫瘤缺氧模式確立後，即腫瘤細胞生長至第45天，體積達500 mm3，即可隨機分組進行實驗。高壓氧氣組 (n=6) 每天給予2.5絕對大氣壓的高壓氧氣90分鐘，共10次，高濃度氧氣組 (n=6) 每天給予1絕對大氣壓的高壓氧氣90分鐘，共10次，控制組 (n=6) 則不給予高壓氧氣處置。每週2次測量腫瘤體積，分別於治療結束第0、14、28天，犧牲老鼠，利用免疫組織化學染色法觀察腫瘤細胞內部缺氧表現與血管分佈情形，並使用西方墨點法觀察Met、VEGF等蛋白質表現。
實驗結果顯示給予高壓氧氣與高濃度氧氣處理後，高壓氧氣組的腫瘤體積在治療結束第0、14、28天皆小於控制組，且達統計上意義 (P<0.05)；而免疫組織染色觀察高壓氧氣組與控制組相比缺氧範圍減少；非缺氧區域，血管分佈明顯增加。因此，利用高壓氧氣治療實體腫瘤，可以改善腫瘤內部缺氧情形，並限制腫瘤體積生長，同時增加血管表現，然而就免疫組織染色觀察Met、VEGF表現則三組間並無明顯差別。

Hyperbaric Oxygen Therapy (HBOT) is the administration of 100% oxygen at atmospheric pressures greater than that at sea level. Hyperbaric oxygen therapy is commonly utilized to treat clinical diseases, such as carbon monoxide poisoning, decompression disease, arterial gas embolism, osteomyelitis, sever anemia, wound healing, and so on. However, malignant disease has been recognized as a contraindication to HBO2 since it might promote residual tumor growth. On the other hand, many studies have described the rapidly growing progression of tumor incurring hypoxia that would be close to the necrotic areas in tumors. Under certain hypoxic condition, tumor cell could become unstable and make gene mutation. Most genes induced by hypoxia are regulated by the hypoxia-induced factor -1 (HIF-1), a protein playing a very important role in tumor development. Vascular endothelial growth factor (VEGF) is also regulated by HIF-1 under transcription lever. Meanwhile, hypoxia activates transcription of the met protooncogene, resulting to higher levels of Met. The overexpression of HIF, VEGF and Met may be able to keep tumor cells proliferation and even metastasis.
The purpose of this study was to establish tumor-hypoxia animal model, and to examine the effect of HBO2 therapy on tumor hypoxia, tumor growth and metastasis. First, we tested hypoxia and monitored the tumor growth separately on the 30th and 45th days after A549 tumor cell were implanted into SCID mice. Next, the tumor implanted mice were treated with NBO and HBO treatment 5 times a week at 2.5 atm absolute for 90 minutes (n=6), then sacrificed on 0, 14, 28 days . Immunohistochemical analysis was used to assist the examination on tumor hypoxia, vasculature expression of VEGF and Met. In the experiment, we have showed the significant differences (p<0.05) in the tumor volume, hypoxia and vasculature between the two growth (Control and HBO2). We concluded that hyperbaric oxygen treatment could decrease tumor hypoxic condition, change the density of tumor blood vessels and limit the growth of tumor. However, the detailed mechanism is still unclear further investigation.

目錄…………………………………………………………………………….I
表次…………………………………………………………………………...Ⅲ
圖次………………………………………………………………………….. IV
中文摘要……………………………………………………………………...Ⅴ
英文摘要……………………………………………………………………...Ⅶ
第一章 緒論
第一節、高壓氧氣治療…………………………………………………….1
第二節、高壓氧氣治療對腫瘤細胞之影響………………………………...5
第三節、腫瘤缺氧理論…………………………………………………….7
第四節、實驗動機…………………………………………………………11
第二章 實驗目的…………….………………………………………………12
第三章 實驗材料與實驗方法
第一節、試劑及抗體…………….…………………………………………13
第二節、緩衝液及SDS-PAGE之備製…………………………………….14
第三節、細胞株的培養……………………………………………………17
第四節、實驗動物…………………………………………………………18
第五節、腫瘤動物模式之建立……………………………………………19
第六節、高壓艙……………………………………………………………19
第七節、實驗分組…………………………………………………………20
壹、控制組
貳、高壓氧氣組
叁、高濃度氧氣組
第八節、實驗參數
壹、腫瘤體積…………………………………………………………...20
貳、腫瘤內部缺氧表現…………………………………………………21
叁、免疫組織化學染色 (Immunohistochemistry)……………………..21
肆、西方點墨法 (Western blot analysis)
一、細胞蛋白質萃取…………………………………………………23
二、蛋白質濃度之定量分析…………………………………………23
三、蛋白質樣本之處理………………………………………………24
四、西方點墨法………………………………………………………24
第九節、數據分析………………………………………………………….25
第四章、實驗結果
第一節、觀察A549細胞株進行異種移植的生長曲線與生長型態…….26
第二節、腫瘤細胞生長至第45天的內部缺氧表現比30天更明顯……26
第三節、經高壓氧氣處理後，限制腫瘤體積生長………………………27
第四節、高壓氧氣治療改善腫瘤缺氧，增加腫瘤內的血管表現...............28
第五節、高壓氧氣治療改善腫瘤缺氧，對VEGF及Met表現的影響...29
第五章 結論………………………………………………………………….31
第六章 討論
第一節、高壓氧氣治療對於腫瘤缺氧及血管生成的影響………………32
第二節、高壓氧氣治療對腫瘤細胞之影響………………………………36
第三節、未來方向…………………………………………………………37
第七章 參考資料…………………………………………………………….72
附錄一………………………………………………………………………...81

1.Alagoz T, Buller RE, Anderson B, Terrell KL, Squatrito RC, Niemann TH, Tatman DJ, Jebson P：Evaluation of hyperbaric oxygen as a chemosensitizer in the treatment of epithelial ovarian cancer in xenografts in mice. Cancer, 75 (9)：2313-22, 1995.
2.Belozerov VE and Van Meir EG：Hypoxia inducible factor-1：a novel target for cancer therapy. Anti-cancer Drugs, 16：901-909, 2005.
3.Brown JM: The hypoxic cell：a target for selective cancer therapy. Cancer Res. 59：5863-70, 1999.
4.Bojes HK, Datta K, XU J, Chin A, Simonianp, Nunn EZ G, and Kehrer JP：Bcl-xL overexpression attenuates glutathione depetion in FL5.12 cells following interleukin-3 withdrawal. Biochem J. 325 (2)：315-319, 1997.
5.Brenner I, Shephard JR and Shek NP：Immune function in hyperbaric environments, diving, and decompression. Undersea Hyperb. Med. 26 (1)：27-39, 1999.
6.Camp RL, Rimm EB, and Rimm, DL：Met expression is associated with poor outcome in patients with axillary lymph node negative breast carcinoma. Cancer. 86：2259-2265, 1999.
7.Chavko M, Braisted JC, Outsa NJ, and Harabin AL：Role of cerebral blood flow in seizures from hyperbaric oxygen exposure. Brain Res. 791：75-82, 1998.
8.Christofori G：New signals from the invasive front. Nature. 441(25)：444-450, 2006.
9.Feldmeier JJ, Onoda JM, Bossung DE, Court WS, and Alecu R：Effect of hyperbaric oxygen on cancer cell metastatic potential and proliferation. Undersea Hyper Med. 24(supl)：130, 1997.
10.Feldmeier JJ, Hampson NB. A systematic review of the literature reporting the application of hyperbaric oxygen prevention and treatment of delayed radiation injuries：an evidence-based approach. Undersea & Hyperbaric Medicine (UHM), 29：4–30, 2002.
11.Feldmeier J, Carl U, Hartmann K, Sminia, P: Hyperbaric oxygen：Dose it promote growth or recurrence of malignancy? Undersea & Hyperbaric Medicine (UHM), 30(1)：1-18, 2003.
12.Ferrara N, Davis-Smyth T：The biology of vascular endothelial growth factor. Endocrine reviews, 18：4-25, 1997.
13.Francis, JT, Griffin LJ, Homer DL, Pezeshkpour HG, Dutka JA and Flynn TE：Bubble-induced dysfunction in acute spinal cord decompression sickness. J Appl. Physiol. 68(4)：1368-75, 1990.
14.Freeman BA, Topolosky MK, and Crapo JD：Hyperoxia increase radical production in rat lung homogenates. Arch Biochem Biophys. 216：477-484, 1982.
15.Folkman J：Tumor angiogenesis: Therapeutic implications. N Engl. J Med., 285：1182-1186, 1971.
16.Folkman J：What is the evidence that the tumors are angiogenesis dependent? J Natl. Cancer Inst., 82：4-6, 1990.
17.Folkman J：Clinical applications of research on angiogenesis. N Engl J Med, 333：1757-1763, 1995.
18.Folkman J：Role of Angiogenesis in Tumor Growth and Metastasis. Seminars in Oncology., 29(6), Suppl. 16 (December)：15-18, 2002.
19.Gale NW, Yancopoulos GD：Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. Gene & development, 13：1055-1066, 1999.
20.Ghoussoub RA, Dillon DA, D’Aquila T, Rimm EB, Fearon ER, and Rimm D L：Expression of c-met is a strong independent prognostic factor in breast carcinoma. Cancer. 82：1513-1520, 1998.
21.Greco O, Marples B, Joiner MC, Scott SD：How to overcome (and exploit) tumor hypoxia for targeted gene therapy. Journal of Cellular Physiology, 197：312-325, 2003.
22.Grim PS, Gottlieb LJ, Boddie A, and Batson E：Hyperbaric oxygen therapy. JAMA. 263: 2216-2220, 1990.
23.Hansbrough JF, Piacentine JG, and Eiseman B：Immunosuppression by hyperbaric oxygen. Surg. 87：662-667, 1980.
24.Hockel M, and Vaupel P：Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J N C I. 93 (4)：266-276, 2001.
25.Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N：Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J. Biol. Chem., 267：26031-26037, 1992.
26.Huang LE, Gu. J, Schau M, and Bunn HF：Regulation of hypoxia-inducible factor l alpha is mediated by an O2-dependent degradation domain via ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. 95：7987-92, 1998.
27.Hunt, TK：Zederfeldt B, Goldtick TK：The Oxygen and Healing. Am. J. Surg. 118：521-5, 1969.
28.Hunt, TK：The physiology of wound healing. Ann Emerg Med. 17(12)：1265-73, 1988.
29.Ito T, Yufu K, Mori A, and Packer L：Oxidative stress alters arginine metabolism in rat brain: effect of sub-convulsive hyperbaric oxygen exposure. Neurochem Int. 29(2)：187-95, 1996.
30.Jain KK：Textbook of hyperbaric medicine. Flagstaff, AZ：Best Publishing, 1996.
31.Jain KK：Textbook of hyperbaric medicine：Indications, contraindications, and complications of hyperbaric oxygen therapy, 103-109, 2004.
32.Johnson RJR, and Lauchlan SC：Epidermoid carcinoma of the cervix treated by 60Co therapy and hyperbaric oxygen. Proceedings of the third international congress on hyperbaric medicine. 648-52, 1966.
33.Kaelin CM, Im MJ, Myers RAM, Manson PN, and Hoopes JE：The effects of hyperbaric oxygen on free flaps in rats. Arch Surg. 125：607-9, 1990.
34.Kallio PJ, Wilson WJ, O’Brien S, Makino Y, and Poellinger L：Regulation of the hypoxia-inducible transcription factor l alpha by the ubiquitin-proteasome pathway. J. Biol. Chem. 274：6519-25, 1999.
35.Kaln J, Krock L, and Piepmeier E：The effect of hyperbaric oxygen on growth and chemosensitivity of metastatic prostate cancer. Anticancer Res. 18：363-7, 1998.
36.Klagsbrun V, D’Amore PA：Vascular endothelial growth factor and its receptors. Cytokine & growth factor reviews, 7(3)：259-270, 1996.
37.Knighton, DR, Halliday B and Hunt TK：Oxygen as an antibiotic. A comparison of the effects of inspired oxygen concentration and antibiotic administration on in vivo bacterial clearance. Arch Surg. 121(2)：191-5, 1986.
38.Kinoshita Y, Kohshi K, Kunugita N, Tosaki T and Yokota A：Preservation of tumour oxygen after hyperbaric oxygenation monitored by magnetic resonance imaging. British Journal of Cancer, 82(1)：88–92, 2000.
39.Lemarie RJ, Hosgood G, VanSteenhouse J, Hodgin EC, Tedford BL, and Strain GM：Effect of hyperbaric oxygen on lipid peroxidation in free skin grafts in rats. Am J Vet Res., 59(7)：913-7, 1998.
40.Li H, Shimura H, Aoki Y, Date K, Matsumoto K, Nalamura T, and Tanaka M：Hepatocyte growth factor stimulates the invasion of gallbladder carcinoma cell lines in vitro. Clin. Exp. Meta. 16：74-82, 1998.
41.Livio Trusolino and Paolo M. Comoglio：Scatter-Factor and semaphoring receptors：cell signaling for invasive growth. Nature Reviews, 2：289-300, 2002.
42.Cejudo-Martin P and Johnson RS：A new notch in the HIF belt: How hypoxia impacts differentiation. Development Cell, 9：575-576, 2005.
43.Marx RE：Radiation injury to tissue. In: Kindwall EP., Whelan HT. (cds) Hyperbaric Medicine Practice (Second Edition). Flagstaff, AZ：Best Publishing Co., 720, 1999.
44.McMillan T, Calhoun KH, Mader JT, Stiernberg CM, Rajarman S：The effect of hyperbaric oxygen on oral mucosal carcinoma. Laryngoscope, 99：241-244, 1989.
45.Menon C, Fraker DL：Tumor oxygenation status as a prognostic marker. Cancer Letters, 221：225–235, 2005.
46.Nakajima M, Sawada H, Yamada Y, Watanabe A, Tatsumi M, and Yamashita J：The prognostic significance of amplification and overexpression of c-met and c-erb B-2 in human gastric carcinomas. Cancer. 85：1894-902, 1999.
47.Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z.：Vascular endothelial growth factor (VEGF) and its receptors. FASEB J, 13：9-22, 1999.
48.Oliner J et al.：Suppression of angiogenesis and tumor growth by selective inhibition of angiopoietin-2. Cancer Cell, 6：507-516, 2004.
49.O’Reilly MS, Holmgren L, Folkman JC. Chen：Angiostatin induces and sustains dormancy of human primary tumors in mice, Nat. Med., 2：689–692, 1996.
50.Oriani G, Marroni A, Wattel F, et al.：Physiology and physiopathology of hyperbaric oxygenation. Handbook on hyperbaric medicine, Springer, 1996, 1-34.
51.Padgaonkar VA, Leverenz VR, Fowler KE, Reddy VN, and Giblin FJ：The effects of hyperbaric oxygen on the crystallins of cultured rabbit lenses: a possible catalytic role for copper. Exp Eye Res. 71(4)：371-83, 2000.
52.Parr C, Watkins G, Mansel RE, and Jiang WG：The hepatocyte growth factor regulatory factors in human breast cancer. Clinical Cancer Res. 10：202-211, 2004.
53.Pennacchietti S, Michieli P, Galluzzo M, Mazzone M, Giordano S, and Comoglio PM：Hypoxia promotes invasive growth by transcriptional activation of the met protoocogene. Cancer Cell, 3：347-61, 2003.
54.Purucker E, and Lutz J：Effect of hyperbaric oxygen treatment and perfluorochemical administration on glutathione status of the lung. Adv Exp Med Biol, 317：131-6, 1992.
55.Rapisarda A, Zalek J, Hollingshead M, Braunschweig T, Uranchimeg B, Bonomi CA, Borgel SD, Carter JP, Hewitt SM, Shoemaker RH, and Melillo1 G：Schedule-dependent inhibitionn of hypoxia-inducible factor-1 protein accumulation, angiogenesis, and tumor growth by Topotecan in U251-HRE glioblastoma xenografts. Cancer Res. 64：6845–6848, 2004.
56.Salceda S, and Caro J：Hypoxia-inducible factor 1  (HIF-1) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. J. Biol. Chem. 272：22642-7, 1997.
57.Semenza GL：Targeting HIF-1 for cancer therapy. Nature Reviews Cancer. 3：721-732, 2003.
58.Sheridan RL, and Shank ES：Hyperbaric Oxygen Treatment：A Brief Overview of a Controversial Topic. J. Trauma. 47：426-35, 1999.
59.Shewell J, and Thompson SC：The effect of hyperbaric oxygen treatment on pulmonary metastasis in the C3H mouse. Eur J Cancer. 16：253-9, 1980.
60.Shi Y, Lee CS, Wu J, Koch CJ, Thom ST, Maity A, Bernhard EJ：Effects of hyperbaric oxygen exposure on experimental Head and Neck tumor growth, oxygenation, and vasculature. Head and Neck, 27：362-369, 2005.
61.Speit G and Bonzheim I：Genotoxic and protective effects of hyperbaric oxygen in A549 lung cells. Mutagenesis 18(6)：545-548, 2003.
62.Sun TB, Chen RL, Hsu YH：The effect of hyperbaric oxygen on human oral cancer cells. Undersea & Hyperbaric Medicine, 31(2)：251-260, 2004.
63.Sugawara J, Fukaya T, Murakami T, Yoshida H, and Yajima A：Hepatocyte growth factor stimulates proliferation, migration, and lumen formation of human endometrial epithelial cells in vitro. Biology of reproduction. 57：936-942, 1997.
64.Suzuki Y, Ono Y, and Hirabayashi Y：Rapid and specific reactive oxygen species generation via NADPH oxidase activation during Fas-mediated apoptosis. FEBS Lett. 425(2)：209-12, 1998.
65.Takiguchi N, Saito N, Nunomura M, Kouda K, Oda K, Furutyama N, Nakajima N： Use of 5-FU plus hyperbaric oxygen for treating malignant tumors: evaluation of antitumor effect and measurement of 5-FU in individual organs. Cancer Chemother. Pharmacol., 47：11-14, 2001
66.Taniguchi K, Yonemura Y, Nojima N, Hirono Y, Fushida S and Fujimura T：The relation between the growth patterns of gastric carcinoma and the expression of hepatocyte growth factor receptor (c-met), autocrine motility factor receptor, and urokinase-type plasminogen activator receptor. Cancer, 82：2112-22, 1998.
67.Thomlinson RH and Gray LH：The histological structure of some human lungcancers and the possible implications for radiotherapy. Br. J. Cancer, 9：539–549, 1955.
68.Tibbles PM and Edelsberg JS：Medical progress: Hyperbaric-oxygen therapy. The N Engl J Med. 334(25)：1642-8, 1996.
69.Trusolino L and Comoglio PM：Scatter-factor and semaphorin receptors: Cell signaling for invasive growth. Nature reviews cancer. 2：289-300, 2002.
70.Ueki T, Fujimoto J, Suzuki T, Yamamoto H, and Okamoto E：Expression of hepatocyte growth factor and its receptor c-met proto-oncogene in hepatocellular carcinoma. Hepatology. 25：862-6, 1997.
71.Veikkola T, Karkkainen M, Lena CW, Alitalo K：Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Research, 60：203-212, 2000.
72.Wang G.L, and Semenza GL：Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem. 270：1230-7, 1995.
73.Zhou J, Schmid T, Schnitzer S, Brüne Bernhard：Tumor hypoxia and cancer progression. Cancer Letters, 1–12, 2005.