학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2013. 2. 송준명.발암 물질에 대한 경각심이 높아지고 있는 현대사회에서 담배, 식품이 함유하는 발암물질에 대한 정량분석 및 그 영향에 대한 연구가 중요해졌다. 본 연구는 reporter gene assay를 기반으로 DNA 손상의 정량분석을 시도하였다. (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)는 담배 및 식품에 함유된 발암물질 이자 돌연변이 유발물질인 benzo[a]pyene의 한 대사체이다. 이 BPDE를 DNA에 직접 처리하여 DNA 돌연변이를 유발하고, DNA 돌연변이에 기인한 단백질 변형에 따른 발현률 변화를 측정하였다. 발암물질 처리를 하는 목표 DNA는 SupF gene으로 원핵세포 뿐 아니라 진핵 세포에도 적용이 가능한 유전자이다. 전체 Plasmid에 돌연변이 유도 물질을 처리하던 기존의 방식과 달리, 음성오류를 제거하기 위해 목표 유전자인 SupF gene(117bp)에만 BPDE를 시간(12-48 h)과 농도 (10-500 μM)를 다르게 처리하였다. 흡광도 측정법에 기초해서 4개의 BPDE-SupF gene Adduct의 농도를 구한 후, 형성된 4가지 종류의 BPDE-SupF gene adduct들을 잔여 셔틀 벡터 (pAcGFP1-N1)와 재 조합하여 4가지 BPDE-SupF gene adduct 포함 재조합 plasmid를 얻었다. 그 결과 원핵세포 뿐 아니라 진핵세포에서도 적용이 가능한 벡터융합체계를 구축하였다. BPDE 처리 조건과 돌연변이가 형성된 SupF-AcGFP1 Fusion plasmid의 단백질 발현률 변화 상관관계를 측정할 수 있는 정량적 조건을 확립하였고, 이를 기반으로 DNA 손상정도의 측정 뿐 아니라 회복률 까지 측정할 수 있는 기반을 마련하였다. 단세포에 직접적으로 형광 이미징 기술을 이용하여 측정을 함으로써, 앞으로 DNA 손상 정도 및 약물 화학물질 독성의 척도로 응용 될 수 있을 것으로 기대된다.국문초록 ··················································································································· ⅰ
목 차 ······················································································································· ⅲ
List of Figures ······································································································· ⅳ
List of Tables ········································································································ ⅴ
List of Abbreviations ··························································································· ⅵ
Ⅰ. 서 론 ·················································································································· 1
Ⅱ. 실험 ······················································································································ 3
1. 실험재료 ············································································································ 3
1-1. 시약 및 용매 ···························································································· 3
1-2. 실험 기구 ·································································································· 4
1-3. 분석기기 ···································································································· 5
2. 실험방법 ············································································································ 6
2-1. Target gene 준비 ··················································································· 7
2-2. Remaining Vector 준비 ······································································ 10
2-3. BPDE처리 ······························································································· 14
2-4. Fusion plasmid vector 재조합 ·························································· 16
2-5. Transfection ··························································································· 17
2-6. 형광이미지 분석 ···················································································· 18
Ⅲ. 결과 ···················································································································· 19
1. SupF PCR ······································································································ 19
2. GFP 제한효소 처리 ······················································································ 21
3. SupF gene BPDE 처리 결과 ····································································· 23
4. BPDE-SupF adduct와 재조합 plasmids 확인 ······································· 25
5. BPDE-SupF adduct 함유한 Fusion vector의 형광 발현 확인 ·········· 27
Ⅳ. 결론 ···················································································································· 34
Ⅴ. 참고문헌 ············································································································ 35
Abstract ··················································································································· 38Maste
