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dc.contributor.advisor김재상-
dc.contributor.author김주송-
dc.creator김주송-
dc.date.accessioned2021-01-25T16:30:14Z-
dc.date.available2021-01-25T16:30:14Z-
dc.date.issued2021-
dc.identifier.otherOAK-000000173047-
dc.identifier.urihttp://dcollection.ewha.ac.kr/common/orgView/000000173047en_US
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/256072-
dc.description.abstractReactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. I identified NADPH oxidase 4 (Nox4) as the major ROS generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using iPS cells generated from Nox4-/- mouse. Differentiation markers showed significantly reduced expression levels indicating the importance of Nox4-generated ROS. From transcriptomic analyses, I found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes. Indeed, addition of IGF2 to culture partly restored the differentiation efficiency of Nox4-/- iPS cells. This results reveal an important signaling axis in control of crucial events in differentiation of pluripotent stem cells mediated by ROS.;활성산소는 줄기세포를 포함한 다양한 세포전달과정에서 이차신호전달물질로서 중요한 역할을 한다. 본 연구를 통해 배양체로부터 삼배엽으로 분화되는 과정에서 NADPH oxidase4(Nox4)가 활성산소를 생산하는 주된 효소라는 것을 확인하였으며, Nox4결핍마우스세포로부터 역분화줄기세포를 만들어 삼배엽분화에서 Nox4의 기능을 확인하였다. Nox4역분화줄기세포로부터 만든 배양체의 삼배엽분화에서 삼배엽분화마커의 유의미한 발현감소는 Nox4에 의한 활성산소가 삼배엽분화에 중요한 역할을 한다는 것을 증명한다. 전사체분석을 통해 배아발생과정에 광범위하게 관여한다 밝혀져 있고 Nox4결핍배양체의 삼배엽분화에서 발현이 크게 감소되어 있는 Insulin-like growth factor2(IGF2)를 Nox4의 하위조절유전자후보로 선택하였다. 실제로 Nox4결핍배양체 삼배엽분화에 IGF2를 넣어줬을 때 삼배엽분화발현이 일부 증가되는 것을 확인할 수 있으며 이러한 결과는 ROS에 의해 매개되는 줄기세포분화의 주요신호전달체계를 이해하는데 큰 도움이 될 것 이라 기대한다.-
dc.description.tableofcontentsINTRODUCTION 1 MATERIALS AND METHODS 3 1. Cell culture 3 2. Production of retroviruses 4 3. Generation of iPS cells 4 4. EB Formation and in vitro differentiation 4 5. Alkaline Phosphatase (AP) Staining 5 6. Coomassie blue Staining 5 7. Immunofluorescence 5 8. RNA Extraction and Quantitative Real-Time PCR 6 9. Immunoblotting analyses 6 10. Apoptosis analysis by flow cytometry 7 11. RNA-Sequencing and bioinformatics analysis 7 RESULTS 10 1. ROS is required for efficient differentiation of EB 10 2. Expression of Nox4 during EB differentiation and generation of Nox4-/- iPS cells 17 3. Generation and functional characterization of Nox4-/- iPS cells 19 4. Transcriptomic analysis of Nox4-/- EB cells 23 5. Nox4 promotes differentiation of three embryonic germ layers via a IGF2-PI3K pathway 28 DISCUSSION 39 REFERENCES 42 국문초록 53-
dc.formatapplication/pdf-
dc.format.extent2264115 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.subject.ddc600-
dc.titleNox4 promotes differentiation of ES cells into derivatives of three embryonic germ layers-
dc.typeDoctoral Thesis-
dc.format.pagev, 54, [1] p.-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 생명과학과-
dc.date.awarded2021. 2-
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