Ocean and Polar Research

Korea Institute of Ocean Science & Technology (한국해양과학기술원)
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Assessment of Immune Parameters of the Wild Pacific Oyster (Crassostrea gigas) using a Flow Cytometry and Neutral Red Retention Assay

유세포 분석기와 Neutral Red Retention Assay를 이용한 자연산 참굴(Crassostrea gigas)의 면역력 측정

  • Hong, Hyun-Ki (School of Marine Biomedical Science (POST BK21) and Marine and Environmental Research Institute, Jeju National University) ;
  • Kang, Hyun-Sil (School of Marine Biomedical Science (POST BK21) and Marine and Environmental Research Institute, Jeju National University) ;
  • Kim, Young-Ok (South Sea Research Institute, KIOST) ;
  • Choi, Kwang-Sik (School of Marine Biomedical Science (POST BK21) and Marine and Environmental Research Institute, Jeju National University)
  • 홍현기 (제주대학교 해양과학대학 해양의생명과학부) ;
  • 강현실 (제주대학교 해양과학대학 해양의생명과학부) ;
  • 김영옥 (한국해양과학기술원 남해연구소) ;
  • 최광식 (제주대학교 해양과학대학 해양의생명과학부)
  • Received : 2012.03.28
  • Accepted : 2012.05.21
  • Published : 2012.06.30
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Abstract

Hemocyte parameters of the wild Pacific oyster Crassostrea gigas inhabiting intertidal zones in small bays (Gwangyang and Jinhae Bay) on the southern coast of Korea were evaluated using flow cytometry and neutral red retention (NRR) assay. Morphological features, cell count, mortality, DNA damage, phagocytosis, and lysosomal membrane stability of hemocytes were analyzed. Three types of hemocytes were identified in the oyster hemolymph: granulocytes, hyalinocytes, and blast-like cells. Immune related functions of hemocyte including phagocytosis and lysosomal membrane stability were significantly different among the study areas (P<0.05), while cell count, mortality, and DNA damage of hemocytes were not significantly different. In Gwangyang Bay, phagocytosis of granulocytes and lysosomal membrane stability of oyster hemocytes inhabiting inside bay were significantly lower than those of oyster hemocytes in outside bay (P<0.05), indicating that oysters in inside bay of Gwangyang were relatively suppressed the immunological function in hemocytes. Contrary to Gwangyang Bay, immune parameters of oyster hemocytes in Jinhae Bay not showed the difference between sampling sites. In conclusion, flow cytometry and NRR assay using oyster hemocyte has a powerful tool to investigate the cell level in a short time due to no-preprocessing of material.

남해의 광양만과 진해만에 서식하는 자연산 참굴의 건강성 평가를 위해 세포성 면역을 담당하는 혈구의 기능들을 유세포 분석기와 Neutral Red Retention(NRR) assay를 이용하여 신속, 정확하게 측정하였다. 광양만과 진해만의 안쪽과 바깥쪽에 서식하는 자연산 참굴의 혈구를 유세포 분석기를 이용하여 형태학적 특성에 따라 혈구의 종류를 분류하고, 혈구 종류별 수, 사멸률, DNA 손상도, 식세포능을 측정하였다. 또한 NRR assay를 이용하여 혈구의 lysosomal membrane stability를 측정하였다. 참굴의 혈구는 granulocytes, hyalinocytes, blast-like cells의 세 가지 종류로 분류되었다. 조사 지역 간의 혈구의 수, 사멸률, DNA 손상도는 유의적 차이가 없었지만, 식세포율과 lysosomal membrane stability와 같은 면역 관련 기능들은 유의적 차이가 있었다. 진해만은 내만과 외만 지역에 서식하는 참굴의 혈구 면역인자들 간에는 유의적 차이가 관찰되지 않았다. 이에 반해, 광양만에 서식하는 참굴은 내만 지역의 섬진대교가 외만 지역의 평산리 지역에 서식하는 참굴보다 낮은 식세포율과 낮은 lysosomal membrane stability를 보여 면역력이 저하되어 있는 것으로 추정된다. 하지만, 해양환경 변화와 시료의 면역력과의 상관관계를 이해하기 위해서는 조사지역의 환경적 특성이나 오염정도, 그리고 시료 내의 오염물질 축적량 등의 객관적인 분석 결과와의 종합적인 고찰이 필요할 것이다. 유세포 분석기와 NRR assay를 이용한 참굴 혈구 집단의 형태 변화 및 면역능 측정 기술은 시료의 전처리 없이 빠른 시간 내에 세포의 특성을 분석할 수 있는 유용한 분석 tool로 활용될 수 있음을 확인하였다.

Keywords

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