Nuclear Medicine and Molecular Imaging

The Korean Society of Nuclear Medicine (대한핵의학회)
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Establishment of a Hepatocellular Carcinoma Cell Line Expressing Dual Reporter Genes: Sodium Iodide Symporter (NIS) and Enhanced Green Fluorescence Protein (EGFP)

나트륨 옥소 공동수송체 유전자와 녹색 형광 유전자의 이중 리포터 유전자를 발현하는 간암세포주 확립

  • Kwak, Won-Jung (Department of Molecular Medicine, Kyungpook National University School of Medicine) ;
  • Koo, Bon-Chul (Department of Physiology, Catholic University of Daegu, School of Medicine) ;
  • Kwon, Mo-Sun (Department of Physiology, Catholic University of Daegu, School of Medicine) ;
  • Lee, Yong-Jin (Department of Nuclear Medicine, Kyungpook National University School of Medicine) ;
  • Lee, Hwa-Young (Department of Molecular Medicine, Kyungpook National University School of Medicine) ;
  • Yoo, Jeong-Soo (Department of Molecular Medicine, Kyungpook National University School of Medicine) ;
  • Kim, Te-Oan (Department of Physiology, Catholic University of Daegu, School of Medicine) ;
  • Chun, Kwon-Soo (Korea Institute of Radiological & Medical Sciences) ;
  • Cheon, Gi-Jeong (Korea Institute of Radiological & Medical Sciences) ;
  • Lee, Sang-Woo (Department of Nuclear Medicine, Kyungpook National University School of Medicine) ;
  • Ahn, Byeong-Cheol (Department of Nuclear Medicine, Kyungpook National University School of Medicine) ;
  • Lee, Jae-Tae (Department of Nuclear Medicine, Kyungpook National University School of Medicine)
  • 곽원정 (경북대학교 의과대학 분자의학교실) ;
  • 구본철 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 권모선 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 이용진 (경북대학교 의과대학 핵의학교실) ;
  • 이화영 (경북대학교 의과대학 분자의학교실) ;
  • 유정수 (경북대학교 의과대학 분자의학교실) ;
  • 김태완 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 전권수 (원자력의학원) ;
  • 천기정 (원자력의학원) ;
  • 이상우 (경북대학교 의과대학 핵의학교실) ;
  • 안병철 (경북대학교 의과대학 핵의학교실) ;
  • 이재태 (경북대학교 의과대학 핵의학교실)
  • Published : 2007.06.30
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Abstract

Purpose: Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. Materials and Methods: A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. Results: The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, $T_{1/2}$ of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. Conclusion: A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene.

목적: 광학과 핵의학 및 자기공명 분자영상 기술은 생체내에서 리포터 유전자의 발현을 비침습적으로 평가할 수 있다. 한가지 이상의 유전자 발현을 영상화 할 수 있는 복합분자영상은 유전자의 발현과 유전자 치료 후 효능의 평가를 다양한 방법으로 반복하여 평가할 수 있다는 장점이 있다. 본 연구에서는 핵의학 영상이 가능한 NIS와 광학 영상이 가능한 EGFP 두가지 유전자를 동시에 발현하는 HepG2-Retro-PNRGW (PGKp-NIS-RSVp-EGFP-WPRE) plasmid를 이용한 간암 세포주(HepG2-NE)를 구축하고, NIS와 EGFP 리포터 유전자의 기능 발현을 체내에서 광학영상과 핵의학 영상으로 확인하고자 하였다. 재료 및 방법: pcDNA-NIS로 부터 NIS 유전자를 분리하여 pRetro-PN vector를 만든 후, pLNRGW (LTR-NeoR-RSV-EGFP-WPRE)로부터 RSV-EGFP-WPRE 조각을 분리하여 최종적으로 NIS와 EGFP 유전자가 동시에 발현할 수 있는 pRetro-PNRGW vector를 구축하였다. 구축된 vector를 이용하여 Retro-PNRGW retrovirus를 생산하였으며, 이를 HepG2 세포에 감염시켜 HepG2-NE 세포주를 만들었다. 이 세포주의 NIS 유전자의 발현은 역전사효소 중합효소 연쇄반응으로 mRNA 발현을 확인하였고, EGFP 유전자의 발현은 형광현미경을 통하여 EGFP 단백질이 발현하는 녹색형광을 관찰함으로써 확인하였다. 이중 리포터 유전자 중 NIS 유전자의 기능은 세포에서 방사능 옥소의 섭취량과 유출량의 측정을 통해서 확인하였다. 이렇게 만들어진 세포를 누드마우스에 이식하여 형광 영상, I-123을 이용한 감마카메라 영상과 I-124를 이용한 소동물용 PET 영상을 획득하였다. 결과: NIS와 EGFP의 이중 리포터 유전자를 가지고 있는 HepG2 세포주가 성공적으로 만들어졌다. 세포의 약 50% 정도가 형광 현미경 아래에서 관찰되었다. NIS 유전자의 발현은 역전사효소 중합효소 연쇄반응 실험을 통해서 확인하였고, NIS가 발현된 세포의 방사능옥소 섭취량은 대조군에 비하여 약 9배 정도 높게 나타났다. 방사능옥소 유출량 실험에서는 약 9분에 반 정도의 옥소가 유출되는 것이 확인되었다. 구축된 세포주를 이식한 후 획득한 형광 영상, 감마카메라과 소동물용 PET 영상에서는 반대쪽의 대조군 세포를 이식한 것에 비하여 뚜렷한 형광신호가 보였고, 더 높은 방사능옥소 섭취가 확인되었다. 결론: NIS와 EGFP의 이중 리포터 유전자를 가지는 간암 세포주가 성공적으로 구축되었고, 소동물에서 두 유전자를 각각 치료용 리포터 유전자와 영상 리포터 유전자로의 사용이 가능할 것이라고 생각된다.

Keywords

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