표제지
목차
국문 요약문 13
Ⅰ. 서론 16
Ⅱ. 문헌 고찰 19
1. FOLFIRI와 근위축 19
1) FOLFIRI의 구성 및 작용기전 19
2) FOLFIRI 치료에 의한 부작용 24
3) 항암화학요법에 의한 근위축 25
4) FOLFIRI 치료로 유도된 근위축 완화 기전 32
2. 당삼 뿌리 유래 Creoside IV 33
1) 당삼 33
2) Creoside IV의 생리 활성 34
Ⅲ. 연구 내용 및 방법 37
1. In vitro : creoside IV가 근원세포 분화 및 FOLFIRI 유도 근위축에 미치는 영향 37
1) 세포실험 연구 디자인 37
2) CCK-8 Assay 38
3) Immunofluorescence 38
4) Real-time quantitative polymerase chain reaction 38
5) Western blot 39
6) 미토콘드리아 막전위 측정 40
7) 미토콘드리아 ROS 측정 40
8) 미토콘드리아 양 측정 40
9) 통계 분석 41
2. In vivo : creoside IV의 zebrafish 모델 내 FOLFIRI 유도 근위축에 미치는 영향 41
1) 동물실험 연구 디자인 41
2) Immunohistochemistry 42
3) 미토콘드리아 손상 정도 확인 42
4) Real-time quantitative polymerase chain reaction 42
5) 통계 분석 43
Ⅳ. 결과 44
1. Creoside IV의 근원세포 분화 촉진 효과 44
1) 세포독성 44
2) 다핵성 MHC-양성 근관 세포 형성 46
3) 근육분화 관련 지표 MyoD, MyoG 및 MHC의 mRNA 발현 49
4) 단백질 합성 관련 지표 Akt, mTOR, p70s6k, FoxO1의 mRNA 발현 54
5) 미토콘드리아 대사 관련 지표 Pgc1α, Sdha, Drp1, Opa1의 mRNA 발현 57
2. Creoside IV의 zebrafish 모델 내 FOLFIRI 유도 근위축 완화 효과 60
1) 근육 표현형 60
2) 근육 분화 관련 지표 myog, smyhc1, myhz2의 mRNA 발현 62
3) 미토콘드리아 표현형 65
4) 미토콘드리아 생합성 관련 지표 pgc1 α tfam의 mRNA 발현 68
5) 미토콘드리아 OXPHOS 관련 지표 sdha, sdhb, atp5fα1 mRNA 발현 71
6) 세포사멸 관련 지표 bcl-2, bax, caspase-9의 mRNA 발현 74
7) 미토콘드리아 다이나믹스 관련 지표 drp1, mfn2의 mRNA 발현 77
3. Creoside IV의 C2C12 근육세포모델 내 FOLFIRI 유도 근위축 완화 효과 80
1) FOLFIRI 및 creoside IV 처리 시간별 MHC, MuRF1의 단백질 발현 80
2) 단백질 합성 관련 지표 Akt, mTOR, p70S6K, MHC 단백질 발현 86
3) 단백질 분해 관련 지표 JNK, FoxO1, MuRF1, MAFbx 단백질 발현 89
4) 미토콘드리아 막전위 92
5) 미토콘드리아 ROS 95
6) AMPK 단백질 발현 98
7) 미토콘드리아 양 101
8) 미토콘드리아 기능 관련 지표 PGC1α, TFAM, DRP1 단백질 발현 104
Ⅴ. 고찰 107
Ⅵ. 요약 및 결론 114
Ⅶ. 참고문헌 117
ABSTRACT 126
Table 1. The number of multinucleated MHC positive cells cells in C2C12 myotubes 47
Table 2. The relative mRNA expression of MyoD and myogenin 50
Table 3. The relative mRNA expression of Myh isoforms 52
Table 4. The relative mRNA expression of Akt, mTOR, p70s6k, and FoxO1 55
Table 5. The relative mRNA expression of Pgc1α, Sdha, Drp1, and Opa1 58
Table 6. The relative mRNA expression of myog, smyhc1, and myhz2 63
Table 7. Mitochondrial morphology in the Tg (mito:EGFP) zebrafish embryo 66
Table 8. The relative mRNA expression of pgc1α and tfam 69
Table 9. The relative mRNA expression of sdha, sdhb, and atp5fα1 72
Table 10. The relative mRNA expression of Bax, Bcl2, and caspase-9 75
Table 11. The relative mRNA expression of drp1 and mfn2 78
Table 12. Protein expressions of MHC during the differentiation period 82
Table 13. Protein expressions of MuRF1 during the differentiation period 84
Table 14. Western blot analysis of Akt, mTOR, p70S6K, and MHC expression in C2C12 cells 87
Table 15. Western blot analysis of JNK, FoxO1, MuRF1, and MAFbx expression in C2C12 cells 90
Table 16. Quantitative analysis of mitochondrial membrane potential in C2C12 cells 93
Table 17. Quantitative analysis of mitochondrial ROS production in C2C12 cells 96
Table 18. AMPK protein expression in C2C12 cells 99
Table 19. Quantitative analysis of mitochondrial mass in C2C12 cells 102
Table 20. Western blot analysis of of PGC1α, TFAM, and DRP1 expression in C2C12 cells 105
Figure 1. Mechanism of action of 5-fluorouracil with luecovorin in cancer cells 21
Figure 2. Mechanism of action of irinotecan 23
Figure 3. The myogenic lineage progression and expression profile of key myogenic modulators during myogenic differentiation 26
Figure 4. Mitochondrial change during skeletal muscle regeneration 29
Figure 5. Mitochondrial dynamics and biogenesis 31
Figure 6. The chemical structure of creoside IV 36
Figure 7. Cell viability of C2C12 cells treated with different concentrations of creoside IV 45
Figure 8. Immunofluorescence staining and number of multinucleated MHC(+) cells in C2C12 myotubes 48
Figure 9. The relative mRNA expression of MyoD and myogenin 51
Figure 10. The relative mRNA expression of Myh isoforms 53
Figure 11. mRNA expression of Akt, mTOR, p70s6k, and FoxO1 56
Figure 12. The relative mRNA expression of Pgc1α, Sdha, Drp1, and Opa1 59
Figure 13. Effects of creoside IV on slow- and fast-twitch muscle fiber in zebrafish embryos 61
Figure 14. The relative mRNA expression of myog, smyhc1, and myhz2 64
Figure 15. Mitochondrial morphology in the Tg (mito:EGFP) zebrafish 67
Figure 16. The relative mRNA expression of pgc1α and tfam 70
Figure 17. The relative mRNA expression of sdha, sdhb, and atp5fα1 73
Figure 18. The relative mRNA expression of Bax, Bcl2, and caspase-9 76
Figure 19. The relative mRNA expression of drp1 and mfn2 79
Figure 20. Protein expressions of MHC during the differentiation period 83
Figure 21. Protein expressions of MuRF1 during the differentiation period 85
Figure 22. Western blot analysis of Akt, mTOR, p70S6K, and MHC expression in C2C12 cells 88
Figure 23. Western blot analysis of JNK, FoxO1, MuRF1, and MAFbx expression in C2C12 cells 91
Figure 24. Representative images and quantitative analysis of mitochondrial membrane potential in C2C12 cells 94
Figure 25. Representative images and quantitative analysis of mitochondrial ROS production in C2C12 cells 97
Figure 26. AMPK protein expression in C2C12 cells 100
Figure 27. Representative images and quantitative analysis of mitochondrial mass in C2C12 cells 103
Figure 28. Western blot analysis of of PGC1α, TFAM, and DRP1 expression in C2C12 cells 106
Figure 29. Effects of creoside IV on FOLFIRI induced muscle atrophy 116