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[표제지 등]=0,1,2

제출문=0,3,1

최종연구보고서 초록=0,4,1

요약문=I,5,6

Summary=VII,11,8

Contents=XV,19,4

목차=XIX,23,4

Figure=XXIII,27,3

Table=XXVI,30,1

그림목차=XXVII,31,3

표목차=XXX,34,1

제1장 연구개발과제의 개요=1,35,1

제1절 목적 및 필요성=1,35,3

제2절 연구개발 범위=3,37,2

제2장 국내ㆍ외 기술개발 현황=5,39,1

제1절 세포성장 및 사멸에 폴리아민의 역할=5,39,9

제2절 Theoredoxin=14,48,3

제3장 연구개발수행 내용 및 결과=17,51,1

제1절 실험재료 및 방법=17,51,1

1. Thioredoxin=17,51,1

1-1. Cell Culture And Transfection=17,51,2

1-2. Chemical Treatments And V-Irradiation=18,52,1

1-3. Cell Growth And Death Measurement=18,52,1

1-4. Chromosome Analysis=18,52,1

1-5. Flow Cytometic And Fluorescence Microscopic Analysis Of The Intracellular Stained Cells=18,52,2

1-6. Western Blot Analysis=19,53,1

1-7. Preparation Of The cDNA Probe And Microarray Hybridization=19,53,1

1-8. Senescence Associated β-Galactosidase (SA-β-Gal) Activity staining=20,54,1

2. SAMDC=20,54,1

2-1. 세포배양=20,54,1

2-2. SAMDC siRNA 제작과 발현=20,54,2

2-3. 폴리아민 농도측정=21,55,1

2-4. 형광을 이용한 세포내 활성산소종수준 측정=21,55,1

2-5. 세포내 Glutathione 수준 결정=21,55,1

2-6. 소핵분석법=22,56,1

2-7. 단세포 전기 영동법=22,56,1

2-8. Western Blot Analysis=22,56,1

2-9. Prporidium Iodide Analysis For Apoptotic Cell Death=22,56,2

2-10. (55)Fe Uptake In SAMDC-Deficient HL-60 Cells(이미지 참조)=23,57,1

2-11. Quantitative Real-Time RT-PCR=23,57,1

2-12. HDAC Adday=23,57,2

3. GAD=24,58,1

3-1. GAD Antisense Construction And Transfection=24,58,1

3-2. Cell Culture=24,58,1

3-3. GAD Activity Assay=24,58,2

3-4. Western Blot Assay=25,59,1

3-5. Propidium Iodide에 Analysis For Apoptotic Cell Death=25,59,1

3-6. 세포내 Glutathione 수준 결정=25,59,1

3-7. Irradiation And H₂O₂=25,59,1

3-8. Microscopy=26,60,1

4. Genistein=26,60,1

4-1. Reagent=26,60,1

4-2. Cell Culture And Cell Growth=26,60,1

4-3. V-Irradiation=26,60,1

4-4. Measurement Of Intercellular ROS Level=27,61,1

4-5. Western Blot Assay=27,61,1

4-6. Morphologic Characteristics Analysis=27,61,1

4-7. Stastistical Analysis=27,61,1

5. ICDH=27,61,1

5-1. Cell Culture And Transfection=27,61,2

5-2. Propidium Iodide Analysis For Apoptotic Cell Death=28,62,1

5-3. Detection Of Reactive Oxygen Species Using Fluorescence Image=28,62,1

6. Thiamine=28,62,1

6-1. Bacterial Strains, Growth Rate, And Cell Survival=28,62,1

6-2. Specific Staining Of Polyacrylamide Gel Electrophoresis And The Measurement Of Mn-SOD Activity=28,62,2

6-3. Nitroblue Tetrazolium (NBT) Reduction Experiment=29,63,1

6-4. 2-Deoxy-D-Ribose Assay For OH Scavenging Activity=29,63,1

제2절 연구결과 및 고찰=30,64,1

1. Theoredoxin=30,64,1

1-1. Thioredoxin Overexpression In The HT-1080 Fibrosarcoma Cells=30,64,15

1-2. Thioredoxin Antisense In The HT-29 Colon Carcinoma Cells=44,78,4

1-3. The Response To Y-Irradiation In The Thioredoxin Antisense-Transfected Cell=47,81,6

2. S-Adenosylmethionine Decarboxylase=53,87,1

2-1. 개요=53,87,2

2-2. Construction Of SAMDC siRNA Expression And Cell Growth=54,88,5

2-3. Intracellular Glutathione, Iron Uptake And Oxygen-Derived Free Radical Level=59,93,2

2-4. Assessment Of DNA Damage=60,94,1

2-5. Histone Modification, DNA Repair Gene And Antioxidant Defense System=60,94,8

2-6. Sensitivity To V-Irradiation In SAMDC-Deficient HL-60 Cells=67,101,8

3. Glutamate Decarboxylase=75,109,1

3-1. 개요=75,109,2

3-2. GAD 67KD Antisense Inhibits The Growth Of HT-29 Colon Carcinoma Cells=76,110,2

3-3. Intracellular Glutathione And Oxygen-Derived Free Radical Level=78,112,1

3-4. Sensitization To Butyrate And pH Reduction=78,112,2

3-5. Sensitization To V-Radiation And H₂O₂=80,114,6

4. Genistein=86,120,1

4-1. 개요=86,120,1

4-2. Effect Genistein On HL-60 Cell Proliferation=86,120,2

4-3. Intercellular ROS Generation By Genistein=87,121,1

4-4. Change Of Intercellular Glutathione Level=87,121,1

4-5. Expression Of Antioxidant Enzyme=87,121,3

4-6. G₂/M Phase Arrest And Apoptosis By Pro-Oxydant Activity Of Genistein=90,124,1

4-7. Expression Of G₂/M Phase Arrest And Apoptosis Related Proteins By Pro-Oxydant Activity Of Genistein=90,124,1

4-8. Different Effect Of-Irradiation Between Human Promyeloid Leukemia HL-60 Cells And Normal Human Lymphocyte=90,124,6

4-9. Difference Of Morphologic Characteristics Between Human Promyeloid Leukemia HL-60 Cells And Normal Human Lymphocyte=95,129,4

5. Isocitrate Dehydrogenase=99,133,1

5-1. 개요=99,133,1

5-2. ICDH Activity=99,133,1

5-3. Intercellular ROS Level=99,133,2

5-4. Sensitization To H₂O₂ And Paraquat=101,135,1

6. Thiamine=102,136,1

6-1. 개요=102,136,1

6-2. Inhibition Of Paraquat-Induced Cell Growth Defect By Thiamaine In A Dose Dependent Manner=102,136,3

6-3. Thiamine Effect On SodA Expression And The Sprcific Activity Of Mn-SOD=104,138,2

6-4. Thiamine Effect On The O₂ Mediated Teduction Of NBT=106,140,1

6-5. OH Scavenging Activity Of Thiamine=106,140,2

제4장 연구개발목표 달성도 및 관련분야 기여도=108,142,1

제1절 연구개발목표 달성도=108,142,1

제2절 관련 분야 기여도=109,143,1

제5장 연구개발결과의 활용계획=110,144,2

제6장 연구개발과정에서 수짐한 해외 과학기술정보=112,146,14

위탁과제: 방사선에 의한 Peroxiredoixn의과산화=125-1,160,1

목차=125-2,161,2

Figure=125-4,163,1

Table=125-5,164,1

요약문=126,165,2

Summary=128,167,2

제1장 국내외 기술개발 현황=130,169,4

제2장 연구개발수행 내용=134,173,1

1. 방사선 조사 및 조직 준비=134,173,1

1-1. 방사선 조사=134,173,1

1-2. 조직 준비 및 분쇄=134,173,1

2. SDS-PAGE 와 2D-PAGE=134,173,1

2-1. SDS-PAGE=134,173,2

2-2. 2D-PAGE=135,174,1

2-3. EZ Staining=136,175,1

3. Immunoblot Analysis=136,175,1

4. In-Gel Trypsinization=136,175,1

4-1. 세척=137,176,1

4-2. 알킬화 반응=137,176,1

4-3. 트립신 처리=137,176,2

5. MALDI-TOF 질량 분광법에 의한 분석=138,177,1

6. 재조합 사람 Prx II C5 정제 및 재조합 사람 Prx II Wild Type 준비=138,177,1

6-1. 단백질 발현 및 세포 배양=138,177,2

6-2. Crude Extract 준비=139,178,1

6-3. Streptomycin Sulfate에 의한 핵산 제거=139,178,1

6-4. 황산 암모늄에 의한 단백질 침전=139,178,1

6-5. Phenyl 소수성 상호작뇽 크로마토그라피=139,178,2

6-6. Bioscale Q2 Column을 이용한 HPLC 음이온 교환 크로마토그라피=140,179,1

6-7. TSK-Heparin 5PW HPLC 크로마토그라피의 이용=140,179,1

6-8. 사람 Prx II Wild Type 농도 및 순도 확인=140,179,1

7. Trx 의존적인 과산화물 분해 활성도 측정=141,180,1

8. Chaperone Activity 측정=141,180,1

9. Prx II C-말만 항혈청과 정제된 단백질 Prx II C5를 이용한 Prx II C5를 인식하는 항원 결정기 제거=141,180,2

10. Immunohistochemistry=142,181,2

제3장 연구수행 결과 및 고찰=144,183,1

1. SDS-PAGE와 Immunoblot을 이용하여 방사선 조사 후 조직별 Prx 분석=144,183,1

2. 2D-PAGE와 Immunoblot을 이용하여 방사선 조사 후 이자 Prx의 과산화 분석=144,183,1

3. Prx II C-말단 항혈청을 이용하여 방사선 조사에 의한 Prx C-말단의 절단 규명=145,184,1

4. 절단된 Prx II의 과산화 정도 분석=145,184,2

5. 방사선 조사에 의해 절단된 Prx II의 잘라진 자리 동정=146,185,2

6. H₂O₂에 의한 Prx의 불활성화 측정=147,186,1

7. Luciferase를 기질로 이용한 Chaperone 활성도 측정=147,186,2

8. Immunogistochemistry를 이옹한 Prx II C5의 이자 조직 내 분포 측정=148,187,19

제4장 연구개발과정에서 수집한 해외 과학기술정보=167,206,5

서지정보양식=171-1,211,1

BIBLIOGRAPHIC INFORMATION SHEET=171-2,212,1

그림목차

Fig 1. Apoptotic Cell Death Of Trx-1 Overexpressed HT-1080 Cells After The Oxidant-Treatments Or Y-Irradiation...(이미지 참조)=32,66,1

Fig 2. The Cell Growth Retardation And Polyploidy Of Trx-1 Overexpressed Cells...(이미지 참조)=34,68,1

Fig 3. The Cell Phase Transition Of Y-Irradiated Trx-1 Overexpressed Cells...(이미지 참조)=36,70,1

Fig 4. The Detection Of The Cellular Senescence Markers In Trx-1 Overexpressed Cells...(이미지 참조)=39,73,1

Fig 5. The Expression Of Trangelin And Tubulin In Trx-1 Overexpressed Cells...(이미지 참조)=40,74,1

Fig 6. Western Blotting Analysis Of Cell Cycle Regulatory Proteins In Trx-1 Overexpressed Cells...(이미지 참조)=42,76,1

Fig 7. G₂/M Phase Peak Time Delay In The DFMO-Treated Cells And In Yhe TRX-Antisense Cells=46,80,1

Fig 8. Radiation-Induced Cell Death In The HT-29 Control Cells And In The DFMO-Trested Cells=46,80,1

Fig 9. Western Blot Analysis Of Trx In The Trx-Antisense Cells And In The DFMO-Treated Cells=48,82,1

Fig 10. Radiation-Induced Cell Death In The HT-29 Cells And In The In The TRX-Antisense Cells=48,82,1

Fig 11. Constructiort Of siRNA Expression Vectors=56,90,1

Fig 12. RT-PCR Analysis Of SAMDC And ODC, Gene Expression In HL-60 Cells Marker (M), Control (C), Empty-Vector Transfectant (CT), SAMDC-Distrupting Transfectant(T)=56,90,1

Fig 13. Induction Of Apoptotic Cell Death In i-SAMDC HL-60...(이미지 참조)=57,91,1

Fig 14. Cell Growth Curve Of HL-60 Cells For 5days...(이미지 참조)=57,91,1

Fig 15. Cell Growth Curve Of SAMDC Disrupting Transfectant HL-60 Cell Treatment With Or Without Desferrioxamine(DEO) luM, N-Acetylcistein (NAC) 100uM=58,92,1

Fig 16. A. Cellular ROS Levels Were Using DCFH-DA Frorescence...(이미지 참조)=61,95,1

Fig 17. (55)Fe Uptake In the i-SAMDC HL-60 Cells=62,96,1

Fig 18. Mean Comet Olive Tail Moment Of HL-60 Cells...(이미지 참조)=62,96,1

Fig 19. AO/EtBr Staining Of HL-60 Cells...(이미지 참조)=63,97,1

Fig 20. RT-PCR Analysis Of Repair Gene Expression In HL-60 Cells...(이미지 참조)=65,99,1

Fig 21. RT-PCR Analysis Of Gluthatione Synthesis-Related Gene In HL-60 Cells=66,100,1

Fig 22. Western Blot Analysis Of Thioredoxirl Expression=66,100,1

Fig 23. Histone H3 And H4 hyperacetylation And Histone Decetylase Activity Measurement In The SAMDC Antisence HL-60 Cells=69,103,1

Fig 24. The Change Of Hypodiploid Contents Produce By Y-Radiation (2Gy) And H₂O₂...(이미지 참조)=70,104,1

Fig 25. The Change Of Hypodiploid Contents Produced By H₂O₂ In The SAMDC Overexpression HT-29 Cells=71,105,1

Fig 26-1. Western Blot Analysis Of GAD6 Expression...(이미지 참조)=77,111,1

Fig 26-2. Cells Stained With Crystal Violet=77,111,1

Fig 26-3. Cell Growth Curves Of HI-29 Cell Lines Using Trypan Blue Exclusion Assay=77,111,1

Fig 27. FACS Artalysis And Fluorescent Images By DCFH-DA=79,113,1

Fig 28. Intrace11ular Total Glutathione Levels Determined By Spectrophotometric Measurement Of Chromophoric Thione Formation (Maximal Absorbance At 400nm) In HT-29 Cell Lines...(이미지 참조)=79,113,1

Fig 29. Microscopic Views Of The HT-29 Cell Lines In The Medium Of Acidic pH...(이미지 참조)=81,115,1

Fig 30-1. The Change Of Hypodiploid Contents Produced By 10Gy Y-Radiation-Treatated In The HT-29 Cell Lines=81,115,1

Fig 30-2. The Change Of Hypodiploid Contents Produced By H₂O₂-Treatated In The HT-29 Cell Lines=82,116,1

Fig 31. The Change Of Hypodiploid Contents Produced By Butyrate-Treatated In The HI-29 Cell Lines=83,117,1

Fig 32. Cell Growth Curve Of Human Promyeloid Leukemia HL-60 Cells...(이미지 참조)=88,122,1

Fig 33. Effect Of Genistein On The Intercellular ROS Level In HL-60 Cells...(이미지 참조)=88,122,1

Fig 34. Effect Of Genistein On The Antioxidant Enzyme Level And Expression In HL-60 Cells...(이미지 참조)=89,123,1

Fig 35. Flow Cytometry Analysis Of Hypodiploid Content And Cell Cycle Distribution Of HL-60 Cells...(이미지 참조)=92,126,1

Fig 35. Effect Of Genistein And N-Acetylcysteine On The Expressions Of G₂/M Phase Arrest (p21) And Apoptosis (Bax) Related Proteins...(이미지 참조)=93,127,1

Fig 37. The Change Of Hypodiploid Contents Produced By Genistein (20uM) And Y-Irradiation (50y) In HL-60 Cells=94,128,1

Fig 38. The Change Of Hypodeploid Contents Produced By Genistein And y-Irradiation In Normal Lymphocytes=96,130,1

Fig 39. ICDH Enzyme Activity Of A549 Cells...(이미지 참조)=100,134,1

Fig 40. H₂O₂ Removing Capacity In The ICDH+ Cells=100,134,1

Fig 41. Hypodiploid Contents In The Control A549 Cells(WT) And ICDH-Overexpressed A549 Cells(ICDH+) Exposed To H₂O₂ And Paraquat=101,135,1

Fig 42. Inhibition Of Paraquat-Induced Damage By Thiamine Treatment In E Coli Cells Exposed To Paraquat...(이미지 참조)=103,137,1

Fig 43. Thiamine (500 mM) Effect On The Mn-SOD Induction By Paraquat Treatment=105,139,1

Fig 44-A. Inhibition Effect Of Thiamine On NBT Reduction Mediated By O₂ Generated In The Xanthine Oxidase/Hypoxanthine System...(이미지 참조)=107,141,1

Fig 44-B. Inhibition Effect Of Thiamine On The Malondiadehyde Formation Of 2-Deoxy-Ribose (1mM) Caused By OH Generated From H₂O After Y-Irradiation (800 Gy, 80 Gy/min)...(이미지 참조)=107,141,1

위탁과제 그림목차

Fig 1. Mammalian Peroxiredoxin Isotypes=151,190,1

Fig 2. Mechanism Of Peroxide Reduction, Hyperoxidation And Retroreduction Of 2-Cys Prx=152,191,1

Fig 3. γ-Irradiation Specifically Induce Truncation Of Prx II In Mouse Pancreas=153,192,1

Fig 4. Hyperoxidation And TruncationOof Mouse Pancreahc Prxs After γ-Irradiation=154,193,1

Fig 5. Truncation Occurs At C-Term Not From Hyper-Oxidized Prx II But From Reduced Prx II=155,194,1

Fig 6. 2D-PAGE Analysis Of C-Term Trumcated Mouse Pancreatic Prx II=156,195,1

Fig 7. Fifth Amino Acid From C-Terminus Of Pancreatic Prx II Is A Site Of Truncation After IR=157,196,1

Fig 8. C-term Truncated Prx II C5 Dose Not Lose Its Peroxidase Activity In The Presence Of High Concentration Of H(2)O(2)(이미지참조)=158,197,1

Fig 9. Prx II C5 Has Higher Chaperone Activity Than Prx II Wt=159,198,1

Fig 10. γ-Irradiation Induced Mouse Pancreatic Prx II C-Term Truncation Is β-Cell Specific=160,199,1

표목차

Table 1. Telative Gene Expression Ratio Of Trx-Overexpressed HT1080 Cells Compared To Control Cells=38,72,1

Tabel 2. Cell Cycle Distribution Of HL-60 Cells After Treationg With Genistein And N-Acedtylcysteine=91,125,1

위탁과제 표목차

Table 1. Characteristics Of Six Isotypes Of Mammalian Peroxiredoxin=149,188,1

Table 2. Sequence Alignment Of Prx II Proteins From Mouse And Human=150,189,1

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