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1 I. 2 II. (ROS) 2. III. 3. (SOD).. (GPx).. 8- (8-OHdG). 8- . AP site (LOOH). (NO). (H2S). IV. 7. SOD Assay Kit -WST. gssg /GSH Quantification Kit Total Glutathione Quantification Kit Biopyrrin EIA Kit Anti-Nitroguanosine antibody -Nucleostain- DNA Damage Quantification Kit -AP Site Counting Liperfluo Spy-LHP. DPPP. NO2/NO3 Assay Kit V. 21. VI. 23. 1. I.. ( ) .. ( ) .. II. (ROS:Reactive Oxygen Species) . (O2) . 1 (O2 -) 2 . (H2O2) H2O2 Fe2+ (3 ) ( OH) . O2 - H2O2 OH (1O2) 4 . (NO ) (NO2 ) (O3) (LOOH) ( 1) .. - - - O2 NO (ONOO ) O2 . (SOD) (H2O2) (O2) H2O2 . (GPx) (H2O) (O2) .. OH. 1 . OH- O2 - e- H2O2 SOD .. OH. O2 O2 - 1 H2O2 H 2O. O2 . NO GSH. NO. NO2 . GPx O3 gssg . LOOH ONOO- H 2O. 1 . 2. III.. (ROS) 2 .. ( ).. 2 .. 3 .. (SOD) . (GPx) .. (DNA) . DNA.

2 (Electron Spin Resonance:ESR) DMPO Carboxy- PTIO .. ( ) . 3. (SOD). SOD O2 - (O2) (H2O2) (Cu/Zn- SOD) (Mn-SOD) . Cu/Zn-SOD . Cu/Zn-SOD . Cu/Zn-SOD 1) . Mn-SOD (TNF)- . (LPS) 2) Mn-SOD .. extracellular-SOD(EC-SOD) . O2 - .. (2H2O2 2H2O + O2).. 2 3).. 34,000 ~ 68,000(U/g ) 413 ~ 500(U/g ). 20,000 ~ 36,000 78 ~ 91. 447 ~ 630 2,600(U/ml). 3 3 ~ 6 . 0 . (GPx). GPx (Se). 21 (Sec) Se . Sec SeH SeOH SeOH 2 . (GSH) SeH ( 3) . GPx 4 . cGPx,GPx1 GIGPx, GPx2 . eGPx,GPx3 PH-GPx, GPx4 4.. GPx (LDL) . eGPx LDL Se . 4) .. 3 . 4.. SH gssg /GSH . GSH . GSH. gssg (Glutathione-S-S-Glutathione) GSH. GSH. GPx H2O2 . GSH H2O2 gssg gssg . gssg gssg . gssg . GSH ( . 3) GSH . GSH GSH .. GSH / gssg 4 . 5) . 6).. 8- (8-OHdG )7). 8-OHdG DNA (dG) 8 . DNA . O O. N N. NH NH. HO. N N. N NH2 ROS N NH2.

3 CH2OH CH2OH. O O. OH OH. deoxyguanosine 8-OHdG. 5 deoxyguanosine . DNA 8-OHdG DNA . 8-OHdG DNA G T . 8-OHdG . 8-OHdG 8) 8-OHdG 2 .. 5. 8- . 8- NO O2 - ONOO- / /.. 8- 8- .. AP Site AP Site DNA apurinic/apyrimidinic site( / ) . AP Site 8-OHdG ( 6) . AP- AP Site . OH . AP Site 9) . AP Site 1 .. AP Site .. AP.. 6 DNA AP Site . DNA 8-OHdG 8- AP Site AP Site . 8-OHdG AP Site 8-OHdG . DNA .. (LOOH).. (Poly-unsaturated fatty acid:PUFA) .. OH H2O. (LH) OH O2. (L ) . LH L LOO LOOH. ( . L . ( . (LOO ) LOO .. LOOH LH.. 7 . 6. (NO). NO NO (NOS) . NO . GMP .. NO . (NO2-, ONOO-) .. (H2S). - (CBS) - (CSE) 3- (3 MST) . (NO) (CO) H2S . (H2O2) .. 10) . H2S pKa (HS-) . HSS- HSSS- (HSn-) (S0) 11) . (-SSH) 12) NO H2O2 . (-SNO) (-SOH) . 13) . SulfoBiotics .. IV.. (1) SOD. S311 SOD Assay Kit-WST.

4 SOD / O2 - . NBT . NBT( ) 100 SOD . SOD Assay Kit-WST . WST-1 100% SOD . - O2 N O3 S SO3- H. N. N. xanthine 2O2 N N. O2 WST-1 formazan xanthine oxidase I. - O2 N O3S SO3- N. N. uric acid +. H2O2 2O2- N N WST-1. Na+. I. SOD O 2 + H 2 O2. inhibition reaction 8 SOD Assay Kit-WST . ( ) . SOD Assay Kit-WST (SOD ) .. 7.. 3). SOD Assay 1) 10 U/ml Kit -WST WST . 2 ~ 3 ml . 2) 0 ~ 4 600 g 10 . 3 Total-SOD .. 3) (2 ~ 3 ml) 10 9720 U/ml of blood ml ml 335 U/ml of blood 15712 U/g( ). 10 ml 0 ~ 4 600 g 10 . 142907 U/g( ).. HeLa 73 U/1 107 cells 4) 0 ~ 4 . HL60 226 U/1 108 cells 600 g 10 2 .. 5) ml ( .. SOD ). 6) 1 ml ml . 7) 15 (10 g). 14). 8) 0 ~ 4 600 g 10 . 1) 10g 400ml .. 9) / ml 2) 5 48 . ml . 3) 48 ( : . % . ( )) (pore size m: ( )) .. 100 .. HeLa: 9 10 cells, HL60 10 cells.

5 6 7. Dilution buffer .. 1) PBS(Phosphate Buffered Saline) 1 ml 2 . 2) PBS . 15).. 3) PBS 1 ml . 4) 0 ~ 4 10,000 g 15 1) . 5) . 50% 100.. Dilution buffer .. 3) . 1) 1) ( 5 ) 4 . 2) 1 . 3) 4 ~ 9 ( mol/l 2) ( . , 10 mmol/l 1 mmol/l ) . EDTA) 3) . ( ) . 4) .. 5) 10,000 g 60 .. 8. (WST ) . WST 50% 20 l SOD 1 (U) . 1U = IC50 = WST 50% . 96 well 1 well 20 l SOD . IC50 . IC50 SOD. SOD .. Kim et al., Antioxidant profile of whole saliva after scaling and root planing in periodontal disease , J. Periodontal Implant Sci., 2010, 40(4), 164 171. Mn-SOD . Mn-SOD 16). KCN Mn-SOD . (1) 20 ml 20 ml Polymorphprep( ) 500 g 35 .. (2) [ mol/l , 10 mmol/l Tris HCl; pH ] ml . 900 g 10 . (3) 5,000 g 10 . (4) [ mol/l , 10 mmol/l Tris HCl; pH ] 50 l . 100,000 g 60 . (5) . (6) 10 mmol/l (Cu/Zn SOD ).

6 (7) SOD Assay Kit-WST . (DDC; 1 mmol/l) Cu/Zn- SOD EC-SOD Mn-SOD 17) .. SOD Assay Kit-WST. M. Miyamura et al., "Difference between Deep Seawater and Surface Seawater in the . Preventive Effect of Atherosclerosis", Biol. Pharm. Bull., 2004, 27(11), 1784-1787 . Y. Sakamoto et al., "The assessment of oxidative stress in infertile patients with - varicocele",BJU Int., 2008, 101(12), 1547-1552. , " .. ", , 2002, 49(10), 679-682. , " .. ", , 2008, 55(12), 640-644. , " ", .. , 2002, 49(1), 25-31.. T. Matsui et al., "Apple procyanidins induced vascular relaxation in isolated rat aorta through NO/cGMP pathway in combination with hyperpolarization by multiple K+ channel activations.", Biosci. Biotechnol. Biochem., 2009, 73(10), 2246-2251. K. Yoshikawa et al., "Novel Phthalide Compounds from Sparassis crispa (Hanabiratake), Hanabiratakelide A-C, Exhibiting Anti-cancer Related Activity", Biol.

7 Pharm. Bull., 2010, 33(8), 1355-1359. 9. , " . ", , 2003, 50(9), 392-398. R. Iida et al., "M-LP, Mpv17-like Protein, Has a Peroxisomal Membrane Targeting Signal COS7 Comprising a Transmembrane Domain and a Positively Charged Loop and Up-regulates ( ) Expression of the Manganese Superoxide Dismutase Gene", J. Biol. Chem., 2003, 278(8), 6301-6306. tat/293 K. Imai et al., "Induction of OGG1 Gene Expression by HIV-1 Tat", J. Biol. Chem., 2005, mTat/293 280(29), 26701-26713. T. Yamamoto et al., "Green Tea Polyphenol Causes Differential Oxidative Environments in Tumor versus Normal Epithelial Cells", J. Pharmacol. Exp. Ther., 2003, 307(1), 230-236 . B. G. Heerdt et al., "The Intrinsic Mitochondrial Membrane Potential of Colonic Carcinoma SW620. Cells Is Linked to the Probability of Tumor Progression", Cancer Res.

8 , 2005, 65 (21), 9861- ( ). 9867. H. R. Rezvani et al., "Hypoxia-inducible Factor-1 , a Key Factor in the Keratinocyte keratinacyte Response to UVB Exposure", J. Biol. Chem., 2007, 282(22), 16413-16422. ( ) H. R. Rezvani et al., "Protective Effects of Catalase Overexpression on UVB-induced Apoptosis in Normal Human Keratinocytes", J. Biol. Chem., 2006, 281(26), 17999-18007. granulosa K. E. Valdez et al., "Regulation of apoptosis in the atresia of dominant bovine follicles of the ( first follicular wave following ovulation", Reproduction, 2005, 130, 71-81. PC12 Z. Wei et al., "Olanzapine Protects PC12 Cells From Oxidative Stress Induced by Hydrogen ( ) Peroxide", J. Neurosci. Res., 2003, 73, 364-368. L. Xiang et al., "Anti-Aging Effects of Phloridzin, an Apple polyphenol, on Yeast via the SOD.)

9 And Sir2 Genes", Biosci. Biotechnol. Biochem., 2011, 75(5), 854-858. A. R. Siafakas et al., "Cell Wall-linked Cryptococcal Phospholipase B1 Is a Source of Secreted Enzyme and a Determinant of Cell Wall Integrity", J. Biol. Chem., 2007, 282(52), 37508-37514. H. Fujita et al., "Reduction of Renal Superoxide Dismutase in Progressive Diabetic Nephropathy", J. Am. Soc. Nephrol., 2009, 20(6), 1303 1313. M. Kitada et al., "Resveratrol Improves Oxidative Stress and Protects Against Diabetic Nephropathy Through Normalization of Mn-SOD Dysfunction in AMPK/SIRT1 Independent Pathway", Diabetes , 2011, 60(2), 634-643. L. Esposito et al., "Reduction in Mitochondrial Superoxide Dismutase Modulates Alzheimer's Disease-Like Pathology and Accelerates the Onset of Behavioral Changes.

10 In Human Amyloid Precursor Protein Transgenic Mice", J. Neurosci., 2006, 26(19), 5167- 5179. T. B. Mysore et al., "Overexpression of Glutathione Peroxidase With Two Isoforms of Superoxide Dismutase Protects Mouse Islets From Oxidative Injury and Improves Islet Graft Function", Diabetes, 2005, 54(7),2109-2116. T. Hasegawa et al., "Suppression of nitrosative and oxidative stress to reduce cardiac . allograft vasculopathy", Am. J. Physiol. Heart. Circ. Physiol., 2009, 296, H1007 H1016. A Hamada et al., "The Effect of Eriobotrya japonica Seed Extract on Oxidative Stress in . Adriamycin-Induced Nephropathy in Rats", Biol. Pharm. Bull., 2004, 27(12), 1961-1964. J. Nishimura et al., "Effect of Fenofibrate on Oxidative DNA Damage and on Gene Expression Related to Cell Proliferation and Apoptosis in Rats", Toxicol.