[보고서]정상세포에서 바이오식품 소재에 의한 분자적 방어기작의 향상에 대한 연구

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정상세포에서 바이오식품 소재에 의한 분자적 방어기작의 향상에 대한 연구
Studies on the improvement of molecular defense mechanism against chemical insults in normal cells by biofood factors 원문보기

보고서 정보
주관연구기관	원광대학교 WonKwang University
연구책임자	김복량
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2011-05
과제시작연도	2010
주관부처	교육과학기술부
사업 관리 기관	한국연구재단
등록번호	TRKO201200002307
과제고유번호	1345126126
DB 구축일자	2013-05-20
키워드	산화적 스트레스,지에스티,햄옥시겐네이즈-1,세포노화,피53,서트1,아플라톡신비1,세포고사oxidative stress,GST,HO-1,senescence,p53,Sirt1,AFB1,apoptosis

초록 ▼

-. Phlorectin, daidzein, equol와 kaempferol 등은 청각세포인 HEI-OC1 세포에서 MAPK와 연결된 Nrf2를 경유하여 HO-1의 발현을 촉진함. 산화적 스트레스를 유도하 청각기능을 손상시키는 cisplatin에 의한 세포고사가 차단되었음.
-. 저농도의 Kaempferol 항산화 효과는 p53 siRNA에 의해 억제되었으나, 고농도의 kaempferol은 자체가 세포고사를 유도하였는데, p53의 작용을 차단한 바 세포사가 오히려 증가하여 Chang 세포 등의 다른 결과와 상이하였음.
-. Sulforaphane과 phlorectin은 간세포 일종인 AML-12세포에서 GST A3, A4, M1, P1 및 T1을 발현시켰음. 이들의 발현이 GSH를 이용한 AFB1-8,9-epoxide의 detoxification 반응을 대사하였는데, Nrf2경로를 통한 GST 활성은 40%정도만 관여하고 있음.
-. Curcumin을 AML-12세포에 투여하면 세포내 GST의 활성을 크게 억제하였으나, GST발현을 야기 시켜서 투여 24시간 후에는 대조군보다 높아짐. GST isozyme들의 발현에는 Nrf2, NF-kB 그리고 AP-1순서로 관여하고 있음. GST A3는 Nrf2가, GST P1은 AP-1과 NF-kB가 크게 관여하였는데 GST T1은 3가지 전사요소의 작용에 무관하였음.
-. 청각세포에서 낮은 농도의 H2O2는 p53/p21활성화를 유도하여 SA-β-galactosidase로 표현되는 세포 노화를 촉진하였는데, luteolin은 Sirt1의 발현을 증가하여서 p53/p21의 활성화를 억제하였음. 이 작용에 세포 노화현상이 감소되었음.

목차 Contents

표지 ... 1
제출문 ... 2
보고서 요약서 ... 3
요약문 ... 4
영문요약문 ... 10
영문목차 ... 13
목차 ... 15
제 1 장 연구개발과제의 개요 ... 17
제 1 절 연구개발의 목적 ... 17
제 2 절 연구개발의 필요성 및 범위 ... 17
제 2 장 국내외 기술개발 현황 ... 24
제 1 절 국내외 기술개발 현황 ... 24
제 3 장 연구개발수행 내용 및 결과 ... 27
제 1 절 이론적, 실험적접근 방법 ... 27
제 2 절 연구내용 및 결과 ... 30
1. 1차년도 (2008) ... 30
(1) 산화적 스트레스에 의한 세포손상을 방지하는 바이오식품소재의 작용기전 연구 ... 30
(2) ARE관련 유전자들의 활성이 산화적 스트레스의 억제효과에 대한 연구 ... 34
(3) 바이오식품소재에 의한 GST 및 GCLC 발현의 신호전달체계 연구 ... 43
2. 2차년도 (2009) ... 46
(1) 산화적 스트레스에 의한 세포손상을 방지하는 바이오식품소재의 작용기전 연구 ... 46
(2) 바이오식품소재에 의한 GST 및 GCLC 발현의 신호전달체계 연구 ... 52
3. 3차년도 (2010) ... 54
(1) 화학적 암발생 과정에서 바이오식품소재의 암예방 효과 연구 ... 54
(2) 바이오식품소재에 항노화 및 암예방 효과 검증 ... 58
제 4 장 목표달성도 및 관련분야에의 기여도 ... 72
제 5 장 연구개발결과의 활용계획 ... 78
제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 80
제 7 장 참고문헌 ... 82

표/그림 (44)

표 Induction of HO-1 expression by phloretin in HEI-OC1 cells.
표 Phloretin increases Nrf2 nuclear translocation.
표 Effects of phloretin on phosphorylations of the MAPKs in HEI-OC1 cells.
표 The protective effect of phloretin on cisplatin-induced apoptosis in HEI-OC1 cells.
표 Preventive effect of phlorertin on the level of Bcl-2 family protein and the activation of capase-8, -9, and -3 by cisplatin in HEI-OC1 Cells
표 Protective effect of kaempferol on cisplatin-induced apoptosis in HEI-OC1 cells. Cells were pretreated with the indicated doses of kaempferol for 18 h and then incubated with 20 μM cisplatin for 30 h.
표 Kaempferol down-regulates the activities of caspase-8, 9 and 3 in HEI-OC1 cells. (A, B and C) Cells were pretreated with indicated doses of kaempferol for 12 h, and incubated with 20 μM cisplatin for 30 h. The enzymatic reactions were analyzed for caspase-8, 9 and 3 activities. Data represent the mean ± S.D. of three independent experiments
표 Induction of HO-1 expression by kaempferol in HEI-OC1 cells
표 Involvement of Nrf2 in the process of HO-1 expression induced by kaempferol
표 The MAPK signal pathway is linked to kaempferol-mediated HO-1 expression
표 Effect of kaempferol on the expression of GCLC and cellular level of GSH in HEI-OC1 cells.
표 Effect of kaempferol on the expression of GCLC and cellular level of GSH in HEI-OC1 cells.
표 Induction of HO-1 expression by daidzein and equol in HEI-OC1 cells.
표 Effects of daidzein and equol on the phosphorylations of MAPKs in HEI-OC1 cells.
표 Protective effects of daidzein and equol on t-BHP-induced cell death in HEI-OC1 cells.
표 CO is involved with the protection of cells against t-BHP-induced cell death.
표 Induction of several isozymes of GSTs and GCLC by SFN and the inhibition of separate GSTs iaozymes and GCLC expression by the treatment of respective siRNA. AML 12 cells were transfected with several siRNAs of GSTs isozymes and GCLC for 24 hours, flowing by treating with 10 μM SFN for 18 hours. The expressions of GSTs isozymes and GCLC were assessed by RT-PCR
표 Cellular senescence causes p53 phosphorylation and p21 expression in HEI-OC1 cells. (Right) Cells were pretreated with luteolin for 12 h and then incubated with 25 μM H2O2 for 3 days. (Right) Cells were incubated with 25 μM H2O2 for 0.5, 1, 3, and 5 days. Western blot analysis was performed using specific antibodies
표 Protective effects of luteolin against H2O2-induced cellular senescence in HEI-OC1 cells. (Left) Cells were incubated with 25 μM H2O2 for 3 days the presence or absence with 10 luteolin. (Right) Cells were incubated with 25 μM H2O2 for 1, 3, and 5 days the presence or absence with 10 luteolin
표 Protective effects of luteolin on H2O2-induced cellular senescence is dependent on p53 phosphorylation and p21 expression. Western blot analysis was performed using specific antibodies.
표 Effects of SIRT1 on H2O2-induced cellular senescence in HEI-OC1 cells.
표 Phloretin decreased the formation of AFBO through its inhibitory effects on CYP1A2 and CYP3A4 activities.
표 Increase of GST activity by phloretin in AML 12 cells. Cells were treated with the indicated concentrations of phloretin for 18 h and were then lysed.
표 Induction of GST isozyme genes by phloretin in AML 12 cells. Cells were treated with the indicated dose of phloretin for 18 h (A) or 20 μM phloretin for the indicated time (B). Then GST isozyme gene expression was measured by RT-PCR. The data show one of three independent experiments
표 The effects of siRNA against GST isozymes on GST activity toward AFBO in AML 12 cells. Cells were transfected with siRNA against GST isozymes. After overnight transfection, cells were treated with 20 μM phloretin for 18 h and were then lysed. The cell lysates were co-incubated with human liver microsomes in the mixtures for AFB1 assay. The data represent the mean±SD of three independent experiments
표 The effects of Nrf2-DN on ARE-luciferase induction, GST genes expression, and GST activity toward AFBO. AML 12 cells were transfected with the indicated amount of Nrf2-DN and/ or ARE plasmid (1 μg). Then cells were treated with 20 μM phloretin for 18 h
표 SFN induced the conjugation potential of AFBO and CDNB with GSH. Cells were treated with the indicated doses of SFN (A) for 12 h. After harvest, cells were sonicated and cell lysates were incubated with 50 μM AFB1, 100 pmol human liver microsomes, 2 mM GSH and an NADPH-generating system. Total GST activity was detected with CDNB (B). MC; mouse cytosol. Data represent the mean±S.D. of three independent experiments.
표 Effect of each GST isozyme on the conjugation potential of AFBO with GSH among SFN-induced GST isozymes
표 Increase of GST activity by curcumin in AML-12 cells. Cells were treated with the indicated dose of curcumin for 24h (A) or 10 μM curumin for the indicated time (B). The cell lysates were incubated with AFB1, human liver microsomes, an NADPH-generating system, and 5 mM GSH. GST activity toward AFBO was detected by HPLC.
표 Effect of curcmin on GST activity and level in AML-12 cells according to the time of treatment. Cells were pretreated with the indicated 5 μM SFN for 12h, and treated with the indicated 10 μM curumin for the indicated time. The cell lysates were incubated with AFB1, human liver microsomes, an NADPH-generating system, and 5 mM GSH. GST activity toward AFBO was detected by HPLC
표 Cucumin induced the expressions of several GST isozymes. Cells were treated with 10 μM curcumin for indicated time. Then GSTs gene expression was measured by RT-PCR.
표 The effects of siRNA against GST isozymes on GST activity toward AFBO in AML 12 cells. Cells were transfected with siRNA against GST isozymes. After overnight transfection, cells were treated with 10 μM curcumin for 24 hour and were then lysed The cell lysates were incubated with AFB1, human liver microsomes, an NADPH-generating system, and 5 mM GSH. GST activity toward AFBO was detected by HPLC.
표 AML 12 cells were transfected with the indicated 0.2μg of Nrf2-DN, NFkB-DN and AP1-DN. Then cells were treated with 10 μM curcumin for 24 h
표 Induction of activities of ARE, NF-kB and AP-1 in HEI-OC1 cells exposed to 10 μM kaempferol and 80 μM H2O2. (A, B and C) Cells were pretreated with or without 80nM p53 siRNA or sirt1 siRNA for 18h, and then incubated with various 10 μM kaempferol for 18 h and then followed by 80 μM H2O2.The luciferase activities of ARE, NF-kB and AP-1 were measured respectively. Data represent the mean ± S.D. of three independent experiments
표 Protective effect of p53 on kaempferol-induced apoptosis in HEI-OC1 cells.
표 p53 protect cell apoptosis by reducing DNA damage and ROS level. (A and B) Cells were pretreated with or without 80 nM p53 siRNA for 18 h, and then incubated with 40 μM and 80 μM kaempferol for 18 h
표 Involvement of JNK expression in kaempferol-induced cell apoptosis in HEI-OC1 cells
표 Effects of p53 on expression of Bcl-2 family proteins and activation of caspase-3 in kaempferol-treated HEI-OC1 cells.
표 Protective effect of p53 on kaempferol-induced apoptosis was not shown in chang cell and AML 12 cell. (A and B) Cells were pretreated with or without 80 nM p53 siRNA for 18 h, and then incubated with 40 μM and 80 μM kaempferol for 18 h in chang cell and AML 12 cell. Cell viability was measured by a MTS assay. Data represent the mean ± S.D. of three independent experiments.
표 Effect of luteolin on cellular senescence in HEI-OC1 cells after H2O2 treatment. (A and B) Cells were incubated with 30 μM H2O2 for 3 days the presence or absence with 2 luteolin. (C) Cells were incubated with 30 μM H2O2 for 1, 3, and 5 days the presence or absence with 2 luteolin
표 Luteolin affects the expression of senescence-associated molecular markers, p53 and p21.
표 Luteolin protects H2O2-induced cellular senescence through SIRT 1 expression.
표 Effect of SIRT 1 gene on H2O2-induced p53 phophorylation and p21 expression in HEI-OC1 cells. Cells pre-incubated for 12 h with medium or luteolin in the absence or presence of siRNA against SIRT 1 gene were exposed to 30 μM H2O2 for 1 day. Western blot analysis was performed using specific antibodies
표 Luteolin prevents DNA from H2O2-induced damage. Cells pre-incubated for 12 h with medium or luteolin in the absence or presence of siRNA against SIRT 1 and p53 gene were exposed to 30 μM H2O2 for 1 day.

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정상세포에서 바이오식품 소재에 의한 분자적 방어기작의 향상에 대한 연구
Studies on the improvement of molecular defense mechanism against chemical insults in normal cells by biofood factors 원문보기

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정상세포에서 바이오식품 소재에 의한 분자적 방어기작의 향상에 대한 연구 Studies on the improvement of molecular defense mechanism against chemical insults in normal cells by biofood factors 원문보기

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