Study of inhibitory effect on cellular growth and angiogenesis of accumulated superoxide by Sephs1 knockout in endothelial cancer cells.

학위논문(석사)--서울대학교 대학원 :자연과학대학 생명과학부,2020. 2. 이병재.Selenophosphate synthetase (SEPHS) is an enzyme that synthesizes selenophosphate which plays as donor for selenoprotein synthesis. In high enkaryotes, there are two isoforms of SEPHS. Only SEPHS2 has enzymatic activity for selenophosphate synthesis, SEPHS1 has no enzymatic activity. However, Sephs1 is an essential gene. knockout of Sephs1 induces embryonic lethality in early mouse embryo and it participates in regulation of redox homeostasis. knockout of Sephs1 in endothelial cancer cell (2H11) attenuated expression of genes which participates in redox homeostasis, and that promoted accumulation of superoxide and decreased bioavailability of nitric oxide in endothelial cancer cells. up-regulated oxidative stress by accumulated superoxide induced DNA damage of cells and inhibited cellular growth via triggering activation of p53 pathway. Accumulated oxidative stress also inhibited cellular motility and attenuated cytoskeleton rearrangement. Sephs1 knockout in endothelial cancer cells inhibited angiogenesis in endothelial cancer cell by down-regulation of NFkB activation and low level of nitric oxide bioavailability.Selenophosphate synthetase (SEPHS)는 selenophosphate를 합성하는 효소로, 셀레늄단백질 합성시 셀레늄 공여자를 합성하는 효소이다. 고등 진핵 생물에는 SEPHS1, 2 두 가지의 형태가 존재하나, SEPHS2만이 이러한 효소로서의 능력을 갖고 있다. 그러나 SEPHS1은 세포 내 산화-환원 항상성을 조절한다. Sephs1의 결손은 혈관 내피 암세포의 산화-환원 항상성에 참여하는 유전자의 발현을 변화시켰고, 이로 인해 세포 내 superoxide의 축적과 과산화수소 및 산화질소의 감소를 유도하였다. 축적된 superoxide는 세포 내 DNA의 손상을 유도하여 p53 단백질을 활성화 시켜 세포의 성장을 저해하였다. 또한 축적된 superoxide에 의한 산화 스트레스는 세포의 이동능력과 세포 골격 조절 기작을 저해하였다. Sephs1 결손에 의해 유도된 이러한 현상은 혈관 내피 세포의 특성인 혈관 신생능력을 저해하였다. Sephs1의 결손에 의한 혈관 신생 저하는 NFkB pathway 활성 저하 및 산화질소의 감소에 의한 결과임이 본 연구를 통해 나타났다.목 차 Ⅰ. 서 론 ·················································· 1 Ⅱ. 재료 및 방법 ········································ 6 1. 재료 ······························································· 6 2. Sephs1 KO 및 KI EC의 확보 ··························· 7 3. Cell culture 및 ROS scavenger 처리 방법 ········ 8 4. Retroviral transduction ·································· 9 5. 활성산소 축적 조사 ··········································9 6. 유세포분석 ····················································· 10 7. PCR 및 qPCR ················································10 8. Westernblot 및 면역염색 ································ 11 9. 세포 성장조사 ··············································· 12 10. 혈관신생조사 ················································ 12 11. 세포의 이동능력조사 ····································· 13 12. 통계처리방법 ················································ 13 Ⅲ. 결 과 ················································· 14 1. Sephs1 KO 및 KI EC의 검증 ·························· 14 2. 활성산소종의 축적을 조절하는 Sephs1의 기능 ··· 19 3. 활성산소 축적의 원인 조사 ···························· 27 4. EC의 성장을 조절하는 Sephs1의 기능················ 35 5. EC의 혈관신생을 조절하는 Sepsh1의 기능 ········ 44 Ⅳ. 고 찰 ·········································· 56 Ⅴ. 참 고 문 헌 ········································ 61 그 림 목 차 그림 1. ······························································ 16 그림 2. ······························································ 17 그림 3. ······························································ 21 그림 4. ······························································ 23 그림 5. ······························································ 25 그림 6. ······························································ 26 그림 7. ······························································ 30 그림 8. ······························································ 31 그림 9. ······························································ 32 그림 10. ···························································· 33 그림 11. ···························································· 37 그림 12. ···························································· 38 그림 13. ···························································· 39 그림 14. ···························································· 40 그림 15. ···························································· 41 그림 16. ···························································· 42 그림 17. ···························································· 43 그림 18. ···························································· 48 그림 19. ···························································· 49 그림 20. ···························································· 50 그림 21. ···························································· 51 그림 22. ···························································· 52 그림 23. ···························································· 53 그림 24. ···························································· 54 그림 25. ···························································· 55Maste