한국발생생물학회지:발생과생식 (Development and Reproduction)

  • 제14권4호
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  • Pages.243-251
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  • 2010
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  • 2465-9525(pISSN)
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  • 2465-9541(eISSN)
한국발생생물학회 (The Korean Society of Developmental Biology)
권호 표지

인간 양수 유래 줄기세포에서 세포투과단백질을 이용한 ESRRB 단백질의 직접도입에 의한 전분화능 관련 유전자의 발현 조절

Up-regulation of Pluripotency-related Genes in Human Amniotic Fluid-derived Stem Cells by ESRRB Conjugated with Cell-Penetrating Peptide

  • 조중현 (차의과학대학교 의생명과학과) ;
  • 이유선 (차의과학대학교 의생명과학과) ;
  • 오미희 (성신여자대학교 생명과학화학부) ;
  • 고정재 (차의과학대학교 의생명과학과) ;
  • 전용필 (성신여자대학교 생명과학화학부) ;
  • 이동률 (차의과학대학교 의생명과학과)
  • Jo, Jung-Hyun (Dept. of Biomedical Science, College of Life Science, CHA University) ;
  • Lee, Yu-Sun (Dept. of Biomedical Science, College of Life Science, CHA University) ;
  • Oh, Mi-Hee (Dept. of Biology, College of Natural Science, Sungshin Women's University) ;
  • Ko, Jung-Jae (Dept. of Biomedical Science, College of Life Science, CHA University) ;
  • Cheon, Yong-Pil (Dept. of Biology, College of Natural Science, Sungshin Women's University) ;
  • Lee, Dong-Ryul (Dept. of Biomedical Science, College of Life Science, CHA University)
  • 투고 : 2010.11.06
  • Accepted : 2010.12.01
  • Published : 2010.12.31
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⟨ 이전 논문 다음 논문 ⟩

Abstract

Estrogen related receptor $\beta$(Esrrb)는 오르판 수용체 중 하나로 전분화능 관련유전자인 Oct4와 Nanog의 발현을 조절함으로써 줄기세포의 미분화를 유지시키고, 지속적인 자기 복제를 가능케 하는 유전자로 알려져 있다. 또한 Feng 등 (2009)은 체세포에 Oct4, Sox2와 함께 Esrrb 유전자를 함께 도입하면, 유전자가 변형된 체세포가 배아 줄기세포와 유사한 유도만능줄기세포로 리프로그래밍(reprograming)되어 진다는 결과를 보고한 바 있다. 본 연구에서는 인간 ESRRB 단백질을 양수유래줄기세포 내로 직접도입하는 방법을 개발하고, 이를 통해 전분화능 관련유전자의 기능 조절을 확인하고자 하였다. 클로닝 된 인간 short-form ESRRB를 세포투과 펩타이드(cell-penetrating peptide, CPP)의 일종인 R7(아르기닌 7개)에 접합(Fusion)하였고, 합성단백질 (R7-ESRRB-His6)의 형태로 배양중인 인간 양수 유래 줄기세포에 처리하여 세포내로 도입하였다. R7-ESRRB-His6 단백질은 5시간 내에 세포막을 통과하였고, 24시간 내에 핵 내로 이동하였다. 또한 핵 내로 이동한 ESRRB 단백질은 OCT4와 NANOG 유전자의 발현을 증가시켰을 뿐만 아니라, 또 다른 전분화능 관련유전자인 SOX2의 발현도 함께 증가시킨다는 것을 확인하였다. 이상의 결과는 세포투과 펩타이드와 유전자의 접합을 통해 생산된 R7-ESRRB-His6 합성단백질이 양수유래줄기세포내로 원활하게 도입되는 것을 확인하였고, 유전자의 변형 없이 전분화능 관련유전자의 기능을 조절할 수 있는 방법임을 확인하였다.

ESRRB (Estrogen related receptor $\beta$) is an orphan receptor, and have a role on maintaining the undifferentiated state and self-renewal of pluripotent stem cell as a transcription factor which regulates the expression of OCT4 and NANOG genes. Also, Feng et al. (2009) reported that Esrrb, Oct4 and Sox2 could induce pluripotent stem cell from somatic cells. The aim of the present study was to develop the direct delivery system of human ESRRB protein into human amniotic fluid-derived stem cells (AFSCs) and to analyze the effect of ESRRB on the regulation of pluripotency-related genes. Human ESRRB has three isoforms arisen by alternative splicing. We cloned short-form ESRRB and made a fusion protein of ESRRB and R7 for an efficient protein transfer to cell. R7 as cell-penetrating peptide(CPP) can help to transfer ESRRB into cells. R7-ESRRB-His6 protein was observed in the cytoplasm and nuclei within 5 hours after treatment. Also, we could observe R7-ESRRB-His6 protein only in the nuclei within 24 hours. Realtime PCR showed that ESRRB increased expression of OCT4 and NANOG as well as SOX2 gene. Therefore, we demonstrated that R7-ESRRB-His6 proteins were efficiently transferred into the nuclei of AFSCs and work well as a possible transcription factor.

Keywords

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