光合菌是一種生長在含水環境的益生菌，大量分佈於各海洋、河川、湖泊、水池和土壤中，細菌因要適應自然界的生存環境、會誘發突變進而產生一些保護性的化合 物。這些化合物被廣泛用於化妝保養品、保健食品及藥物應用中。光合益生菌的化合物具有延緩器官老化、美白功能、抑制腫瘤生長及轉移、調節血脂、抗氧化能 力、免疫功能調節的優異功效。這與細胞內含豐富的類胡蘿蔔素有極大的關聯性。利用Rhodobacter sphaeroides WL-APD 911萃取物(lycogen)中其組合物，ζ-胡蘿蔔素(ζ-carotene)、四氫蕃茄紅素(neurosporene)、甲氧基四氫蕃茄紅素 (methoxyneurosporene)、球形烯醇(spheroidenone)四種成分，研究證明其抗氧化及去除自由基能力優於茄紅素且對細胞刺 激性更低，更擁有降低巨噬細胞產生分泌的一氧化氮(NO)和激活一氧化氮合成酶(iNOS)的表達。Lycogen同時具有降低因化療藥品順鉑 (cisplatin)引起的腎損傷及延長存活率。炎症性腸病(Inflammatory Bowel Disease, IBD)是常見的自身免疫性疾病。在針對小鼠葡聚醣硫酸鈉(dextran sulphate sodium, DSS)誘發的結腸炎，口服lycogen可減少發炎因子白細胞介素-1β (IL-1β)及腫瘤壞死因子-α (TNF-α)的激活現象。同時，lycogen顯著延長有嚴重結腸炎小鼠的存活率，可用來改善各種炎症誘導的傷害，lycogen更具有抑制MMPs造 成膠原蛋白降解的能力，也具下調NF-κB活性，輔助抗癌藥物抑制血管增生，更同時具有抑制癌細胞轉移之功效，這些結果確定了lycogen是一種新的生物材料。

Photosynthetic bacteria are one kind of probiotics growing in aquatic environment and often found in the oceans, rivers, lakes, ponds and soil. In order for bacteria to adapt to their natural environment, they accumulate mutation and produce some protective compounds. These compounds are widely used in cosmetic products, healthy food supplements and other pharmaceutical applications. The compounds produced by photosynthetic bacteria have excellent effects on anti-aging, skin whitening, inhibiting tumor growth and metastasis, regulating blood lipids, antioxidant capacity, and regulating immune functions. These effects are related to the abundant carotenoids present in bacteria. Indeed, research has proven that four components, ζ-carotene, Neurosporene, Methoxyneurosporene and Spheroidenone from Rhodobacter sphaeroides WL-APD 911 (lycogen) are better than lycopene in the antioxidant capacity and removing free radicals, and they are less irritating to cells. Furthermore, they are able to reduce NO secretion and iNOS expression in macrophages. Lycogen can reduce the kidney damage caused by the chemotherapy drug, Cisplatin, and extend the survival rate. Inflammatory bowel disease (IBD) is a common autoimmune disease. In murine model of dextran sulphate sodium (DSS)-induced colitis, oral administration of lycogen can reduce the activation of IL-1β and TNF-α. The significant increase in the survival rate in mice with severe colitis suggests that it can improve a variety injuries induced by inflammation. Moreover, lycogen is able to inhibit collagen degradation cased by MMPs, reduce NF-κB activity, suppress angiogenesis when combined with other anti-cancer drugs, and prevent cancer cell metastasis. Therefore, these effects above identify lycogen as a new biomaterial.

論文審定書…………………………………………………………………………….i
誌謝…………………………………………………………………………………....ii
中文摘要……………………………………………………………………………...iii
Abstract……………………………………………………………………………....iv
目錄……………………………………………………………………………...........v
圖目錄……………………………………………………………………………......vi
表目錄………………………………………………………………………………..xii
一、前言………………………………………………………………………………1
二、實驗材料與方法…………………………………………………………….…18
三、研究結果………………………………………………………………….……48
四、討論………………………………………………………………….…………77
五、表……………………………………………………………………….………83
六、圖片….………………………………………………………………………....95
七、參考文獻………...……………………………………………………………189
簡歷……………………...…………………………………………………………199

Adler V, Yin Z, Tew KD, Ronai Z. Role of redox potential and reactive oxygen species in stress signaling. Oncogene 1999;18:6104-11.

Afonso V, Champy R, Mitrovic D, Collin P, Lomri A. Reactive oxygen species and superoxide dismutases: role in joint diseases. Joint Bone Spine 2007;74:324-9.

Albelda S M, Smith CW, Ward PA. Adhesion molecules and inflammatory injury. FASEB 1994;8:504-12.

Albrecht M, Klein A, Hugueney P, Sandmann G, Kuntz, M. Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating zeta-carotene desaturation. FEBS Lett 1995;372:199-202.

Baillie JK, Thompson AA, Irving JB, Bates MG, Sutherland AI, MacNee W, Maxwell SR, Webb DJ. Oral antioxidant supplementation does not prevent acute mountain sickness: double blind, randomized placebo-controlled trial. QJM 2009;102:341-8.

Belkhiri A, Richards C, Whaley M, McQueen SA, Orr FW. Increased expression of activated matrix metalloproteinase- 2 by human endothelial cells after sublethal H2O2 exposure. Lab Invest 1997;77:533-9.

Berger AC, Alexander HR, Tang G, Wu PS, Hewitt SM, Turner E, Kruger E, Figg WD, Grove A, Kohn E, Stern D, Libutti SK. Endothelial monocyte activating polypeptide II induces endothelial cell apoptosis and may inhibit tumor angiogenesis. Microvasc Res 2000;60:70-80.

Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. JAMA 1997;297:842-57.

Bukowska B. 2,4,5-T and 2,4,5-TCP induce oxidative damage in human erythrocytes: the role of glutathione. Cell Biol Int 2004;28:557-63.

Carr A, Frei B. Does vitamin C act as a pro-oxidant under physiological conditions? FASEB J 1999;13:1007-24.

Chang WW, Liu JJ, Liu CF, Liu WS, Lim YP, Cheng YJ, Lee CH. An extract of Rhodobacter sphaeroides reduces cisplatin-induced nephrotoxicity in mice. Toxins 2013;5:2353-65.

Choudhari SM, Ananthanarayan L, Singhal RS. Use of metabolic stimulators and inhibitors for enhanced production of beta-carotene and lycopene by Blakeslea trispora NRRL 2895 and 2896. Bioresour Technol 2008;99:3166-73.

Christopherson KS, Bredt DS. Nitric oxide in excitable tissues: physiological roles and disease. J Clin Invest 1997;100:2424-9.

Davies KJ. Oxidative stress: The paradox of aerobic life. Biochem Soc Symp 1995;61:1-31.

Duarte TL, Lunec J. Review: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C. Free Radic Res 2005;39: 671-86.

Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005;366:1736-43.

Fang FC. Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide related antimicrobial activity. J Clin Invest 1997;99:2818-25.

Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4-25.

Feskens EJ, Virtanen SM, Räsänen L, Tuomilehto J, Stengard J, Pekkanen J, Nissinen A, Kromhout D. Dietary factors determining diabetes and impaired glucose tolerance. A 20-year follow-up of the finnish and Dutch cohorts of the seven countries study. Diabetes Care 1995;18:1104-12.

Fidler IJ, Gersten DM, Hart IR. The biology of cancer invasion and metastasis. Adv Cancer Res 1978;28:149-250.

Folkman J. Clinical applications of research on angiogenesis. N Engl J Med 1995;333:1757-63.

Folkman J. What is the evidence that the tumors are angiogenesis dependent? J Natl Cancer Inst 1990;82:4-6.

Francis FJ. Carotenoids as food colorants. Cereal Foods World 2000;45:198-203.

Fraser AG, Debnam ES, Dhillon AP, Pounder RE. Gastric epithelial cell proliferation and histological damage after hypertonic sodium chloride: the effect of variation in the strain of rat. Int J Exp Pathol 1992;73:241-50.

Fukumura D, Gohongi T, Kadambi A, Izumi Y, Ang J, Yun CO, Buerk DG, Huang PL, Jain RK. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Natl Acad Sci USA 2001;98:2604-9.

Galiano RD, Tepper OM, Pelo CR, Bhatt KA, Callaghan M, Bastidas N, Bunting S, Steinmetz HG, Gurtner GC. Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am J Pathol 2004;164:1935-47.

Gamaley IA, Klyubin IV. Roles of reactive oxygen species: signaling and regulation of cellular functions. Int Rev Cytol 1999;188:203-55.

Giavazzi R, Valoti G, Ferri C, Nicoletti MJ. Preclinical models to evacuate angiogenesis inhibitors. In “Relevance of Tumor Models for Anticancer Drug Development”. H. H. Fiebig and A. M. Burger, Eds. Karger, Basel. 1999;54:157-68

Glauser MP, Heumann D, Baumgartner JD, Cohen J. Pathogenesis and potential strategies for prevention and treatment of septic shock: an update. Clin Infect Dis 1994;18:S205-16.

Griffioen AW, Molema G. Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev 2000;52:237-268.

Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal 2001;13:85-94.

Hailman E, Vasselon T, Kelley M, Busse LA, Hu MC, Lichenstein HS, Detmers PA, Wright SD. Stimulation of macrophage and neutrophils by complexes of lipopolysccharide and soluble CD14. J Immunol 1996;156:4384-90.

Hamed S, Ullmann Y, Masoud M, Hellou E, Khamaysi Z, Teot L. Topical erythropoietin promotes wound repair in diabetic rats. J Invest Dermatol. 2010;130:287-94.

Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest 1997;100:2153-7.

Hazzalin CA, Le Panse R, Cano E, Mahadevan LC. Anisomycin selectively desensitizes signalling components involved in stress kinase activation and fos and jun induction. Mol Cell Biol 1998;18:1844-54.

Hengartne MO. The biochemistry of apoptosis. Nature 2000;407:770-6.

Huang ZZ, Li H, Cai J, Kuhlenkamp J, Kaplowitz N, Lu SC. Changes in glutathione homeostasis during liver regeneration in the rat. Hepatology 1998;27:147-53.

Hunter CN, Hundle BS, Hearst JE, Lang HP, Gardiner AT, Takaichi S, Cogdell RJ. Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides. J Bacteriol 1994;176:3692-7.

Hyun, JY, Myoung EM, Haeng SP, Suhn YI, Young HK. Chitosan oligosaccharide (COS) inhibits LPS-induced inflammatory effects in RAW 264.7 macrophage cells. Biochem Biophys Res Commun 2007;358:954-9.

Jha P, Flather M, Lonn E, Farkouh M, Yusuf S. The antioxidant vitamins and cardiovascular disease. A critical review of epidemiologic and clinical trial data. Ann Intern Med 1995;123:860-72.

Jia G, Cheng G, Gangahar DM, Agrawal DK. Involvement of connexin 43 in angiotensin II-induced migration and proliferation of saphenous vein smooth muscle cells via the MAPK-AP-1 signaling pathway. J Mol Cell Cardiol 2008;44:882-90.

Jin J, Kwon YW, Paek JS, Cho HJ, Yu J, Lee JY, Chu IS, Park IH, Park YB, Kim HS, Kim Y. Analysis of differential proteomes of induced pluripotent stem cells by protein-based reprogramming of fibroblasts. J Proteome Res 2011;10:977-89.

Jones SA, O'Donnell VB, Wood JD, Broughton JP, Hughes EJ, Jones OT. Expression of phagocyte NADPH oxidase components in human endothelial cells. Am J Physiol 1996;271:H1626-34.

Joubert J, Malan SF. Novel nitric oxide synthase inhibitors: a patent review. Expert Opin Ther Pat 2011;21:537-60.

Kamata H, Shibukawa Y, Oka SI, and Hirata H. Epidermal growth factor receptor is modulated by redox through multiple mechanisms. Effects of reductants and H2O2. Eur J Biochem 1999;267:1933-44.

Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997;9:240-6.

Khachik F, Beecher GR, Goli MB, Lusby WR, Daitch CE. Separation and quantification of carotenoids in human plasma. In “Methods in Enzymology”. Packer L, Ed. New York: Academic Press 1992;213:205-19.

Kim YM, Jang JW, Lee OH, Yeon J, Choi EY, Kim KW, Lee ST, Kwon YG. Endostatin inhibits endothelial and tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase. Cancer Res 2000;60:5410-3.

Kukereja RC, Hess ML. The oxygen free radical system: form equations through membrane-protein interactions to cardiovascular injury and protection. Cardiovasc Res 1992;26:641-55.

Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication. Int J Biochem Cell Biol 2004;36:1171-86.

Li H, Fu X, Zhang L, Huang Q, Wu Z, Sun T. Research of PDGF-BB gel on the wound healing of diabetic rats and its pharmacodynamics. J Surg Res 2008;145:41-8.

Liaudet L, Soriano FG, SzabÓ C. Biology of nitric oxide signaling. Crit Care Med 2000;28:N37-52.

Liotta LA, Rao CN, Barsky SH. Tumor invasion and the extracellular matrix. Lab Invest 1983;49:636-49.

Liu WS, Chen MC, Chiu KH, Wen ZH, Lee CH. Amelioration of dextran sodium sulfate-induced colitis in mice by Rhodobacter sphaeroides extract. Molecules 2012;17:13622-30.

Liu WS, Kuan YD, Chiu KH, Wang WK, Chang FH, Liu CH, Lee CH. The extract of Rhodobacter sphaeroides inhibits melanogenesis through the MEK/ERK signaling pathway. Mar Drugs 2013;11:1899-908.

MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Ann Rev Immunol 1997;15:323-50.

Madigan S, Bakermans-Kranenburg M, van Ijzendoorn M, Moran G, Pederson D, Benoit D. Unresolved states of mind, anomalous parental behavior, and disorganized attachment: a review and meta-analysis of a transmission gap. Attach Hum Dev 2006;8:89-111.

Morazzani M, de Carvalho DD, Kovacic H, Smida-Rezgui S, Briand C, Penel C. Monolayer versus aggregate balance in survival process for EGF-induced apoptosis in A431 carcinoma cells: Implication of ROS-P38 MAPK-integrin alpha2beta1 pathway. Int J Cancer 2004;110:788-99.

Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM. Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 2000;18:1135-49.

Oda Y, Saito K, Yamauchi H, Mori M. Lactic acid fermentation of potato pulp by the fungus Rhizopus oryzae. Curr Microbiol 2002;45:1-4.

Park BK, Zeng X, Glazer RI. Akt1 induces extracellular matrix invasion and matrix metalloproteinase-2 activity in mouse mammary epithelial cells. Cancer Res 2001; 61:7647-53.

Padro T, Ruiz S, Bieker R, Bürger H, Steins M, Kienast J, Büchner T, Berdel WE, Mesters RM. Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia. Blood 2000;95:2637-44.

Paul A, Pendreigh RH, Plevin R. Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages. Br J Pharmacol 1995;114:482-8.

Paust J. Recent progress in commercial retinoids and carotenoids. Pure Appl Chem 1991;63:45-58.

Peus D, Beyerle A, Vasa M, Pott M, Meves A, Pittelkow MR. Antipsoriatic drug anthralin induces EGF receptor phosphorylation in keratinocytes: requirement for H2O2 generation. Exp Dermatol 2004;13:78-85.

Poon Ronnie TP, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Onco 2001;19:1207-25.

Pradhan L, Nabzdyk C, Andersen ND, LoGerfo FW, Veves A. Inflammation and neuropeptides: the connection in diabetic wound healing. Expert Rev Mol Med 2009;11:e2.

Qureshi ST, Gros P, Malo D. The LPS locus: Genetic regulation of host respones to bacterial lipopolysaccharide. Inflamm Res 1999;48: 613-20.

Roper MB, Ladha JK. Biological nitrogen fixation by heterotrophic and phototrophic bacteria in association with straw. Plant Soil 1999;174:211-24.

Salonen JT, Nyyssonen K, Salonen R. Fish intake and the risk of coronary disease. N Engl J Med 1995;333:937

Salonen JT, Nyyssonen K, Tuomainen TP, Mäenpää PH, Korpela H, Kaplan GA, Lynch J, Helmrich SP, Salonen R. Increased risk of non-insulin dependent diabetes mellitus at low plasma vitamin E concentrations: a four year follow up study in men. BMJ 1995;311:1124-7.

Schumann RR, Pfeil D, Lamping N, Kirschning C, Scherzinger G, Schlag P, Karawajew L, Herrmann F. Lipopolysaccharide induces the rapid tyrosine phosphorylation of the mitogen-activated protein kinases erk-1 and p38 in cultured human vascular endothelial cells requiring the presence of soluble CD14. Blood 1996;87:2805-14.

Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996;10:709-20.

Sener M, Hsin J, Trabuco LG, Villa E, Qian P, Hunter CN, Schulten K. Structural model and excitonic properties of the dimeric RC-LH1-PufX complex from Rhodobacter sphaeroides. Chem Phys 2009;357:188-97.

Shi J, Maguer L. Lycopene in tomatoes: Chemical and physical properties affected by food processing. Crit Rev Food Sci Nutr 2000;40:1-42.

Shimokata H, Muller DC, Fleg JL, Sorkin J, Ziemba AW, Andres R. Age as independent determinant of glucose tolerance. Diabetes 1991;40:44-51.

Sies H. Oxidative stress: Oxidants and antioxidants. Exp Physiol 1997;82:291-5.

Siwik DA, Colucci WS. Regulation of matrix metalloproteinases by cytokines and reactive oxygen/nitrogen species in the myocardium. Heart Fail Rev 2004;9:43-51.

Stadtman ER. Role of oxidant species in aging. Curr Med Chem 2004;11:1105-12.

Stohs SJ, Bagchi D. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 1995;18:321-36.

Sweet MJ, Hume DA. Endotoxin signal transduction in macrophages. J Leuk Biol 1996;60:8-26.

Thomas HE, Darwiche R, Corbett JA, Kay TW. Interleukin-1 plus γ-interferon-induced pancreatic β-cell dysfunction is mediated by β-cell nitric oxide production. Diabetes 2002;51: 311-6.

Vacca A, Ribatti D, Presta M, Minischetti M, Iurlaro M, Ria R, Albini A, Bussolino F, Dammacco F. Bone marrow neovascularization, plasma cell angiogenic potential and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma. Blood 1999;93:3064-73.

Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44-84.

Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998; 93:411-22.

Wang CC, Liu WS, Chang FH, Tsai PY, Tsai MK, Shiang JC, Wen ZH. Rhodobacter sphaeroides extract improves glucose homeostasis in streptozotocin-induced diabetic mice. J Microb Biochem Technol 2014;6:038-42.

Wang CY, Liu HJ, Chen HJ, Lin YC, Wang HH, Hung TC, Yeh HI. AGE-BSA down-regulates endothelial connexin 43 gap junctions. BMC Cell Biol 2011;12:19-30.

Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835-70.

Wu WT, Liu WS. Anti-inflammatory property of biomaterial carotenoids production by Rhodobacter sphaeroidese WL-APD911. Adv Mater Res 2011;287-290: 2028-31.

Yang TH, Lai YH, Lin TP, Liu WS, Kuan LC, Liu CC. Chronic exposure to Rhodobacter sphaeroides extract lycogen™ prevents UVA-induced malondialdehyde accumulation and procollagen I down-regulation in human dermal fibroblasts. Int J Mol Sci 2014;15:1686-99.

Yasuda M, Ohzeki Y, Shimizu S, Naito S, Ohtsuru A, Yamamoto T, Kuroiwa Y. Stimulation of in vitro angiogenesis by hydrogen peroxide and the relation with Ets-1 in endothelial cells. Life Sci 1999;64:249-58.

Yu JA, Ye YJ, Lin ZX. Effect of carotenoids in photosynthetic bacteria on inhibiting lipid per-oxidation. Shanghai Traffic Univ 1998;32:107-9.

Zhang W, QU Y, Pretlow TP, Li B. Gene cloning and genomic expression analysis from ACF-A study on pre-colon cancer. In “Cancer Genetics and Tumor suppressor genes”. Cold Spring Harbor Laboratory, New York, USA. 2004:234.