형광 흥분세포분리를 이용한 추정 윤부상피줄기세포의 분리

• 영문명: Isolation of Putative Limbal Epithelial Stem Cells by Fluorescein Activated Cell Sorting
• 발행기관: 대한안과학회
• 저자명: 김미금 신경선 정경아 신기철 위원량 이진학 이재림 박기숙 손영숙
• 간행물 정보: 『대한안과학회지』Ophthalmological Society,volume46,number3, 521~527쪽, 전체 7쪽
• 주제분류: 의약학 > 기타의약학
• 파일형태: PDF
• 발행일자: 2005.03.31

영문 초록

Purpose: To analyze the isolating pattern of slow cycling cells as putative limbal epithelial stem cells (PLESCs) using Hoechst exclusive cell sorting. Methods: Rabbits were injected with 5-bromo-2-deoxyuridine (Brd U) 1 month prior to be sacrificed. After obtaining limbal tissues, fluorescence-activated cells were sorted on a Coulter EPICS 753 after they had been incubated with Hoechst 33342 and propidium iodide. Two different methods were applied to sort PLESCs. Side-population(Sp) cells were obtained using gates with dichroic mirror to detect low Hoechst blue and red after verapamil was treated. Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 BP filter. Brd U-retaining cells were counted and their sizes were evaluated in each gated sample to compare isolating pattern of PLESCs in each method. Results: The percentages of Sp cells and of the Hoechst negative fraction were 0.96±0.79% and 16.01±13.60%, respectively(p=0.021). Homogeneity and density of the small cells were higher in Hoechst negative fraction than in Sp cells. The percentage of Brd U-retaining cells was 47.36±10.34% and 47.14±14.94% in Sp cells and Hoechst negative fraction, respectively(p>0.05), and they were 10 times higher than in non-Sp and Hoechst positive fraction(p=0.000). Conclusions: Hoechst negative exclusion without verapamil more efficiently isolated PLESCs than Sp did.

키워드

Brd U-retaining cell, Cornea, Epithelium, Hoechst exclusion, Side population