Clinical and Experimental Pediatrics

The Korean Pediatric Society (대한소아청소년과학회)
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Single Cell Dissociation Methods for Flow Cytometric Cell Death Analysis of Hypoxia-Ischemia Injured Newborn Rat Pup Brain

저산소성 허혈성 뇌손상이 유발된 신생백서에서 단일세포의 분리

  • Hwang, Jong Hee (Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine) ;
  • Sung, Dong Kyung (Samsung Biomedical Research Institute) ;
  • Choi, Chang Won (Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine) ;
  • Kang, Saem (Samsung Biomedical Research Institute) ;
  • Chang, Yun Sil (Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine) ;
  • Park, Won Soon (Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine) ;
  • Lee, Munhyang (Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine)
  • 황종희 (성균관대학교 의과대학 삼성서울병원 소아과) ;
  • 성동경 (삼성생명과학연구소) ;
  • 최창원 (성균관대학교 의과대학 삼성서울병원 소아과) ;
  • 강샘 (삼성생명과학연구소) ;
  • 장윤실 (성균관대학교 의과대학 삼성서울병원 소아과) ;
  • 박원순 (성균관대학교 의과대학 삼성서울병원 소아과) ;
  • 이문향 (성균관대학교 의과대학 삼성서울병원 소아과)
  • Received : 2004.11.22
  • Accepted : 2005.01.16
  • Published : 2005.05.15
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Abstract

Purpose : Newborn brain tissue has to be dissociated into a single cell suspension for flow cytometric analysis of cell death during hypoxia-ischemia. Thus the development of a method to dissociate cells from the brain tissue with least damage and maintenance of membrane and antigen integrity remains the challenge for the in vivo application of this technique. We evaluated the efficacy of mechanical or enzymatic (collagenase or tryspin) methods of brain tissue disaggregation. Methods : The extent of the damage to the plasma membrane and loss of the characteristics of the membrane induced with each dissociation method was determined by comparing the flow cytometric results labeled with both fluorescent annexin V and propidium iodide of the newborn rat pup brain tissue in the control group (n=10) and in the 48-hour after hypoxia-ischemia group (n=10). Results : In the control group, the cell percentage of damaged, apoptotic and necrotic cells of both hemispheres with the mechanical dissociation method was significantly increased compared to the trypsin or collagenase method. In the 48-hour after hypoxia-ischemia group, the cell percentage of apoptotic and necrotic cells of the right hemisphere with the collagenase method significantly increased, and live cells significantly decreased compared to the left hemisphere, control group. Although the same trend was observed, the extent of alterations made with the trypsin method was significantly less compared to the collagenase method. Conclusion : The dissociation of neonatal brain tissue for flow cytometric analysis with collagenase was most efficacious with the least cell damage and preservation of the plasma membrane characteristics.

목 적: 저산소성 허혈성 손상을 받은 신생아의 뇌조직에서 유세포 방법을 통해 세포의 사멸을 분석하기 위해서는 단일세포의 분리가 이루어져야 한다. 본 연구는 세포 분리에 있어서 세포막의 소실을 최소화하고 항원성을 유지하기 위하여 물리적인 방법과 효소 처리를 통한 세포 분리방법의 효율성에 대해 알아보고자 하였다. 방 법 : 생후 7일된 10마리의 SD 신생백서에 우측 경동맥 결찰 후, 8% 산소에 노출시켜 저산소성 허혈증의 손상을 유발하였으며 48시간이 지난 후 뇌조직을 얻어 같은 수의 정상 대조군과 비교하였다. 세포 분리는 물리적인 방법(pipette)과 효소 처리(trypsin 및 collagenase) 방법을 통하여 이루어 졌으며, 세포막의 손상 정도와 범위에 대해서는 annexin V 및 propidium iodide의 형광 염색을 통한 유세포 분석방법을 이용하였다. 결 과 : 정상 대조군에서, 물리적인 방법을 통한 세포 분리가 양반구 모두에서 효소 처리를 한 경우에 비해서 세포의 사멸과 괴사가 통계적으로 유의하게 증가하였다. 저산소성 허혈증을 유발한 군 중, collagenase를 이용하여 세포 분리를 시행한 경우에서 우측 반구의 세포 사멸과 괴사의 비율이 좌측 반구 및 정상 대조군 보다 유의하게 증가하였다. 효소 처리를 통한 세포 분리에서는 서로 유사한 경향을 보였으나, trypsin을 이용한 경우가 collagenase를 이용한 경우에 비해 세포 변화의 정도가 유의하게 감소하였다. 결 론 : 신생아의 뇌조직에서 collagenase를 이용한 단일 세포 분리방법이 세포막의 손상을 최소화하면서 세포막의 성상을 보존할 수 있는 가장 유용한 방법이었다.

Keywords

Acknowledgement

Supported by : Samsung Biomedical Research Institute

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