예쁜꼬마선충을 이용한 화합물 독성 평가법 구축 연구
초록/요약
Toxicological evaluation is essential for the development of drugs and the risk assessment of environmental toxicants. Here, the chemical toxicological evaluation method was established by using a model nematode, Caenorhabditis elegans, and 3T3-L1 normal murine cells. This method is rapid, convenient, and cost-effective and lacks animal ethics issues as compared to the conventional toxicity test system using mammalian animals. In this study, topoisomerase II inhibitors, etoposide and irinotecan are selected as model chemicals because they are used for the clinical anticancer drugs and potential environmental toxins. To evaluate the chemical toxicity, the growth and reproduction of C. elegans were observed, and cellular toxicity in 3T3-L1 cells was investigated. Etoposide significantly retarded the growth, egg laying rate, and egg hatching rate in C. elegans. Etoposide injured reproductive tissue of C. elegans, so decreased the number of gonad cells and induced the enlargement of gonad cell nuclei. Etoposide potently inhibited the proliferation of 3T3-L1 cells, with IC50 values of 37.8 ± 7.3 and 9.3 ± 1.6 μM after 24 and 48 hr of treatment, respectively, via the induction of cell cycle arrest at the G2/M phase and apoptotic cell death. Irinotecan, the other topoisomerase inhibitor had less reproductive toxicity in C. elegans, as compared to etoposide. Irinotecan did not decrease the number of eggs laid in C. elegans as compared to control, while etoposide significantly decreased the number of eggs laid. The IC50 values of irinotecan were 237.1 ± 86.9 and 93.5±85.4 μM after 24 and 48 hr of treatment, respectively, and these value are much higher than etoposide. These experimental system may be useful to study mechanism underlying the side effects of various chemicals including topoisomerase inhibitors.
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Abstract···························································································i
List of Figures··················································································iii
1. 서론·························································································01
2. 재료 및 방법··············································································06
2.1. 예쁜꼬마선충(C. elegans) 배양··················································06
2.2. 시약····················································································06
2.3. 균주의 준비········································································08
2.4. NGM 배지 제조법·································································08
2.5. 예쁜꼬마선충(C. elegans)의 성장속도 측정·································09
2.6. 예쁜꼬마선충(C. elegans) 생식능력 측정····································09
2.6.1. 예쁜꼬마선충(C. elegans)의 산란능력 측정···························09
2.6.2. 예쁜꼬마선충(C. elegans)의 알 부화능력 측정······················10
2.7. 예쁜꼬마선충(C. elegans) 생식선 세포(gonad germ cell) 관찰··········10
2.8. 3T3-L1 세포주 배양 조건과 배양방법······································11
2.9. 세포독성 측정·····································································12
2.10. 3T3-L1 세포의 형태와 주기 분석···········································12
2.10.1. 3T3-L1 세포의 형태 관찰···············································13
2.10.2. 3T3-L1 세포의 핵 형태 관찰··········································13
2.11. 통계분석············································································13
3. 결과 및 고찰·············································································15
3.1. Etoposide가 예쁜꼬마선충에 미치는 영향···································15
3.1.1. Etoposide가 예쁜꼬마선충의 성장에 미치는 영향··················15
3.1.2. Etoposide가 예쁜꼬마선충의 생식능력에 미치는 영향············18
3.2. Etoposide가 3T3-L1 정상쥐세포에 미치는 영향··························30
3.2.1. Etoposide가 3T3-L1 세포의 생존에 미치는 영향··················30
3.2.2. Etoposide가 3T3-L1 세포의 형태변화에 미치는 영향·············33
3.2.3. Etoposide가 3T3-L1 세포의 핵 크기에 미치는 영향··············37
3.2.4. Etoposide가 3T3-L1의 세포의 주기에 미치는 영향················41
3.3. Irinotecan이 예쁜꼬마선충에 미치는 영향·································44
3.3.1. Irinotecan이 예쁜꼬마선충의 성장에 미치는 영향·················44
3.3.2. Irinotecan이 예쁜꼬마선충의 생식능력에 미치는 영향···········48
3.4. Irinotecan이 정상쥐세포 3T3-L1 세포에 미치는 영향··················53
4. 고찰 및 결론·············································································55
5. 참고문헌···················································································59
