Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Organ Induction by Combined Dose of bFGF and HGF in Animal Cap Assay of Early Xenopus laevis Embryos.

Xenopus laevis 초기 배의 동물극 분리배양에서 bFGF와 HGF 혼합처리에 의한 기관유도

  • 진정효 (창원대학교 자연과학대학 생물학과) ;
  • 윤춘식 (창원대학교 유전공학연구소) ;
  • 이호선 (창원대학교 자연과학대학 생물학과) ;
  • 박용욱 (창원대학교 자연과학대학 생물학과) ;
  • 정선우 (창원대학교 자연과학대학 생물학과)
  • Published : 2004.06.01
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Abstract

Fibroblast growth factors (FGFs) are known to induce multiple functions in early development, including mesoderm formation, gastrulation movement and antero-posterior patterning. The induction of mesoderm from Xenopus presumptive ectoderm and the combination effect on inducing organs of bFGF(basic FGF) and HGF (Hepatocyte Growth Factor) were studied. Explants were cultured in the combined solution for 3 days to normal embryo arrive at St. 43. These effects on combined dose were examined by histological experiment and by immunohistochemical method. The concentrations of growth factors were tested in 0, 0.5, 1, 10 and also tested in 50 ng/ml of bFGF, and 0, 1, 10, 50 and 100ng/ml of HGF respectively. The synergistic effects were seen in the combined-dose of bFGF and HGF rather than in single dose. Various organs were differentiated and highest inducing effects were seen at the combined concentration of 1 ng/ml of bFGF and 10ng/ml of HGF, and at the concentration 10ng/ml of bFGF and 1 ng/ml of HGF. The bFGF induces various organs from cultured animal cap explants and the effects are time and dose-dependent. HGF is also a potent mitogen for renal tubular cells and for mature hepatocytes in primary culture. Eyes were developed in high percentage at the combined concentration of 1 and 10ng/ml of bFGF, and 1 and 10 ng/ml of HGF. From the induced eye and normal embryonic eye, RPE65 was commonly detected by monoclonal antibodies 40All and 25F5 and the localization of RPE65 was seen by AP reaction.

FGFs는 Xenopus의 초기 배발생에서 중배엽 형성, 전후축패턴형성, scatter factor로서 낭배기의 기관형성에 관여하는 등 다양한 기능을 가지고 있는 것으로 알려져 있다 그 중 bFGF는 배양 분리편으로부터 다양한 기관을 유도해낼 수 있으며 그 효과는 처리시간 및 농도 의존적이라고 알려져 왔다. 본 연구는 Xenopus의 예정표피역을 분리하여 bFGF와 HGF을 단독 및 복합처리 하였을 때 기관분화 및 유도효과를 검토하기 위하여 실시하였다. 단독처리 및 복합 처리된 배양액에 동물극 분리편을 정상배가 st. 43에 이를 때까지 2$0^{\circ}C$에서 3일간 배양하여 조직학적 및 면역조직화학적 방법으로 조직의 분화양상을 확인하였다. 성장인자는 bFGF를 0, 0.5, 1. 10. 50 ng/ml의 농도와 HGF를 0, 1, 10, 50, 100ng/ml의 농도로 조합하여 처리한 결과 bFGF 단독처리 때보다 HGF와의 혼합처리에서 기관분화율의 상승효과가 관찰되었다. 분화된 기관은 1 ng/ml의 bFGF 와 10ng/ml의 HGF, 10ng/ml의 bFGF와 1ng/ml의 HGF의 농도에서 매우 다양한 것으로 나타났다. 눈은 1과 10ng/ml의 bFGF ,그리고 1과 10 ng/ml의 HGF 농도조합에서 높은 비율로 분화하였다. 또한 분리편 배양에 의해 유도된 눈과 정상 배의 눈에서 RPE65를 인식하는 단일클론 항체 40All, 25F5를 사용하여 AP 반응이 강하게 나타나 눈의 유도를 확인할 수 있었다.

Keywords

References

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