생명과학회지 (Journal of Life Science)

  • 제23권2호
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  • Pages.167-174
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  • 2013
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

항산화반응을 유발하는 물질의 검색에 적용할 수 있는 형질전환 마우스 생산을 위한 새로운 Prx 6/Luc 벡터시스템의 제조 및 폐암세포주에서 반응성 확인

Construction and In vitro Study of a Prx 6/Luc Vector System for Screening Antioxidant Compounds in the Transgenic Mice

  • 이영주 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 남소희 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 김지은 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 황인식 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이혜련 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 최선일 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 곽문화 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이재호 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 정영진 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 안범수 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 황대연 (부산대학교 생명자원과학대학 바이오소재과학과)
  • Lee, Young Ju (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Nam, So Hee (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Kim, Ji Eun (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Hwang, In Sik (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Hye Ryun (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Choi, Sun Il (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Kwak, Moon Hwa (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Jae Ho (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Jung, Young Jin (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • An, Beum Soo (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Hwang, Dae Youn (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University)
  • 투고 : 2012.06.01
  • 심사 : 2013.01.16
  • 발행 : 2013.02.28
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초록

Peroxiredoxin 6 (Prx 6)는 티올-특이적 항산화 단백질에 속하는 효소로서 산화적 스트레스로부터 세포를 보호할 뿐만 아니라 과산화물의 환원작용을 촉매한다. 본 연구에서는 인간 Prx 6의 promoter를 이용하여 항산화반응을 유발하는 추출물을 효과적으로 스크리닝하는 새로운 형질전환마우스를 개발하는 최종목적을 달성하기 위한 중간단계로서, hPrx 6/Luc 벡터를 개발하고, 이들 벡터의 안정적 발현과 성공적 반응성을 세포주를 이용하여 확인하고자 하였다. 이를 위해, 인간 Prx 6 promoter를 증폭하여 luciferase cDNA와 결합한 hPrx 6/Luc 벡터를 제조하였으며, 제조된 벡터를 제한효소 절단과 염기서열분석을 통해 확인하였다. hPrx 6/Luc 벡터는 NCI-H460 세포에 transfection한 후 인삼(KWG), 홍삼(KRG), 맥문동(LP), 홍문동(RLP)의 4가지 추출물을 처리하여 luciferase activity를 측정하였다. 그 결과, luciferase activity는 4가지 추출물에 의해 효과적으로 증가하였고, 특히 KRG과 LP를 처리한 그룹이 KWG과 RLP를 처리한 그룹보다 높았다. 또한, luciferase activity는 RLP 농도에 의존적으로 증가하였다. hPrx 6/Luc 벡터와 hPrx 6 mRNA반응의 차이를 비교하기 위해, 4가지 추출물을 처리한 후 hPrx 6 mRNA의 양을 RT-PCR로 분석하였다. 그러나, hPrx 6 mRNA의 양은 비록 고농도의 RLP 추출물에서는 약간의 증가가 관찰되었지만, 대조군에 비하여 4가지 추출물에서 유의적인 차이가 없었다. 한편, 4가지 추출물에 의한 superoxide dismutase (SOD) 활성의 변화는 비록 일부 차이는 있었지만 hPrx 6/Luc 벡터와 유사한 반응을 나타내었다. 따라서, 이러한 결과는 hPrx 6/Luc 벡터는 성공적으로 제조되었고, 세포내에서 안정적으로 발현하면서 항상화물질에 민감하고 정량적으로 반응할 수 있음을 제시하고 있다. 더불어, 이러한 세포주에서 확인결과를 바탕으로 형질전환마우스가 개발된다면, 항산화물질을 정량적으로 스크리닝하는 시스템으로 적용가능성이 매우 높음을 보여주고 있다.

Peroxiredoxin 6 (Prx 6) is a member of the thiol-specific antioxidant protein family, which may play a role in protection against oxidative stress and in regulating phospholipid turnover. The aim of this study was to determine whether a human Prx 6/Luc vector was stably expressed and responded to antioxidants in a lung cell line (NCI-H460). To achieve this, the luciferase signal, hPrx 6 mRNA expression, and superoxide dismutase (SOD) activity were measured in transfectants with a hPrx 6/Luc plasmid after treatment with four antioxidant extracts, including Korea white ginseng (KWG), Korea red ginseng (KRG), Liriope platyphylla (LP), and red Liriope platyphylla (RLP). First, the hPrx 6/Luc plasmid was successfully constructed with DNA fragments of human Prx 6 promoter, amplified by PCR using genomic DNA isolated from NCI-H460 cells, and cloned into the pTransLucent reporter vector. The orientation and sequencing of the hPrx 6/Luc plasmid were identified with restriction enzyme and automatic sequencing. A luciferase assay revealed significant enhancement of luciferase activity in the four treatment groups compared with a vehicle-treated group, although the ratio of the increase was different within each group. The KRG- and LP-treated groups showed higher activity than the KWG- and RLP-treated groups. Furthermore, the luciferase activity against RLP occurred roughly in a dose-dependent manner. However, the level of endogenous hPrx 6 mRNA did not change in any group treated with the four extracts. The SOD activity was in agreement with the luciferase activity. Therefore, these results indicate that the hPrx 6/Luc vector system may successfully express and respond to antioxidant compounds in NCI-H460 cells. The data also suggest that the Prx 6/Luc vector system may be effectively applied in screening the response of hPrx 6 to antioxidant compounds in transgenic mice.

키워드

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