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
1
조회
0
다운로드
3
인용
0
참고문헌
키워드
초록
Estrogen related receptor β(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 내로이동한 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.
Abstract
ESRRB (Estrogen related receptor β) 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.
참고문헌 (16)
- Bombail V, "Estrogen receptor related beta is expressed in human endometrium throughout the normal menstrual cycle", Hum Reprod, 23, pp.2782~2790 (2008)
- Chen X, "Integration of external signaling pathways with the core transcriptional network in embryonic stem cells", Cell, 133, pp.1106~1117 (2008)
- Feng B, "Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb", Nat Cell Biol, 11, pp.197~203 (2009)
- Frankel AD, "Cellular uptake of the tat protein from human immunodeficiency virus", Cell, 55, pp.1189~1193 (1988)
- Giguère V, "Identification of a new class of steroid hormone receptors", Nature, 331, pp.91~94 (1988)
- Kim D, "Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins", Cell Stem Cell, 4, pp.472~476 (2009)
- Lee JE, "Evaluation of 28 human embryonic stem cell lines for use as unrelated donors in stem cell therapy : implications of HLA and ABO genotypes" (2010), Cell Transplant
- O’Malley J, "New strategies to generate induced pluripotent stem cells", Curr Opin Biotech, 20, pp.1~6 (2009)
- Onishi A, "The orphan nuclear hormone receptor ERRbeta controls rod photoreceptor survival", Proc Natl Acad Sci USA, 통권 : 107, pp.11579~11584 (2010)
- Takahashi K, "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors", Cell, 126, pp.663~676 (2006)
- van den Berg DL, "Estrogen-related receptor beta interacts with Oct4 to positively regulate Nanog gene expression", Mol Cell Biol, 28, pp.5986~5995 (2008)
- Xie W, "Constitutive activation of transcription and binding of coactivator by estrogen-related receptors 1 and 2", Mol Endocrinol, 13, pp.2151~2162 (1999)
- Yu S, "Orphan nuclear receptor estrogen-related receptor-beta suppresses in vitro and in vivo growth of prostate cancer cells via p21(WAF1", CIP1) induction and as a potential therapeutic target in prostate cancer, 2008, pp.27, 3313~3328
- Zhang X, "Esrrb activates Oct4 transcription and sustains self-renewal and pluripotency in embryonic stem cells", J Biol Chem, 283, pp.35825~35833 (2008)
- Zhou H, "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins", Cell Stem Cell, 4, pp.381~384 (2009)
- Zhou W, "Identification and characterization of two novel splicing isoforms of human estrogen-related receptor beta", J Clin Endocrinol Metab, 91, pp.569~579 (2006)