KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Fluorescence Detection for Protoporphyrin IX Induced from 5-ALA and ALA-methyl ester in Incubated Liver Cancer Cells

간암 세포주에서 5-ALA 및 ALA-methyl ester에 의해 유도된 Protoporphyrin IX의 형광 검출

  • Kim, Myung-Hwa (Department of Chemistry, Keimyung University) ;
  • Kim, Jung-Mi (Department of Chemistry, Keimyung University) ;
  • Kim, Hyun-Jeong (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • Lee, In-Seon (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • Kim, Kyung-Chan (Department of Digital Physics, Keimyung University) ;
  • Lee, Chang-Seop (Department of Chemistry, Keimyung University)
  • 김명화 (계명대학교 화학과) ;
  • 김정미 (계명대학교 화학과) ;
  • 김현정 (계명대학교 전통미생물자원개발 및 산업화연구센터) ;
  • 이인선 (계명대학교 전통미생물자원개발 및 산업화연구센터) ;
  • 김경찬 (계명대학교 디지털물리학과) ;
  • 이창섭 (계명대학교 화학과)
  • Published : 2007.06.30
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Abstract

To clarify the usefulness of fluorescent diagnosis for cancer, we investigated the optimal method of administrating 5-aminolevulinic acid (5-ALA), 5-aminolevulinic acid methyl ester (ALA-methyl ester) by analyzing fluorescence signal of Protoporphyrin IX (PpIX) in the cultured normal and cancer cells. 5-ALA and ALA-methyl ester was injected as a photosensitizer to the cancer liver cells (HepG2) and normal liver cells (Chang). Chang and HepG2 cells were incubated with various concentrations of 5-ALA and ALA-methyl ester (0-800 ${\mu}g/mL$). The accumulation of PpIX induced by 5-ALA and ALA-methyl ester was in HepG2 and Chang. The cell viability was measured by MTT assay. Fluorescence of PpIX in HepG2 cell was excited at a wavelength ($\lambda$ = 410 nm) and showed an emission spectrum at 603.2 nm, 660.8 nm and 603.2 nm, 661.4 nm which could be related to the PpIX generation induced by the applied 5-ALA and ALA-methyl ester, respectively. The experimental results showed that fluorescence signal of PpIX was proportional to the concentration of 5-ALA and ALA-methyl ester in tumor cells, but measured with low concentration in normal cells. Another results showed that the PpIX formation rate induced by ALA-methyl ester is higher than that of 5-ALA.

형광을 이용한 암 진단을 위해 배양된 정상 및 암세포주에 광민감제인 5-ALA, ALA-methyl ester를 투여하고 세포 내 외에서 생성된 Protoporphyrin IX (PpIX)의 형광을 측정하여 5-ALA, ALA-methyl ester 투여의 최적 농도를 조사하였다. 정상 간세포주 (Chang) 및 간암 세포주 (HepG2)에 5-ALA와 ALA-methyl ester를 농도별로 투여하여 5-ALA와 ALA-methyl ester에 의해 유도된 PpIX의 생성을 확인하고, MTT assay로 세포생존율을 측정하였다. 배양된 cell에 5-ALA와 ALA-methyl ester를 투여한 후 24시간 동안 배양함으로써 생성되는 PpIX의 양은 형광의 강도로 측정하였다. 이 때의 형광 (emission) 스펙트럼은 여기 파장이 410 nm일 때 603.2 nm, 660.5 nm와 603.2 nm 및 661.4에서 형광 봉우리가 관찰되었다. PpIX의 형광 강도를 측정한 결과, PpIX는 정상세포에서는 낮은 농도로 축적이 되는 반면에 암세포에서 더 높은 농도로 축적되었으며, 세포 외보다는 세포 내에서 더 높은 농도로 축적됨을 알 수 있었다. 또한 5-ALA 및 ALA-methyl ester에 의한 PpIX의 생성에 대한 형광 강도는 ALA-methyl ester가 5-ALA보다 더 큰 것으로 관찰되었다.

Keywords

References

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