CAJ | 학술논문

目的比较不同的分离介质对小鼠原代肝星状细胞(hepatic stellate cells,HSCs)分离纯化效率的差异.方法肝门静脉原位灌注,预灌注不含钙镁离子的D-Hanks液及含有Ⅳ型胶原酶的灌注液,取消化后的肝脏,分别采用Ficoll-Paque PLUS、Percoll、Nycodenz作为分层介质,密度梯度离心纯化原代HSCs.显微镜下观察细胞纯度并计算细胞得率.锥虫蓝染色判断细胞活率.激光共聚焦显微镜观察原代HSCs的固有荧光情况.油红O染色观察获得原代HSCs的纯度情况.结果采用Ficoll-Paque PLUS、Percoll、Nycodenz分离细胞得率分别为(1±0.5)×106,(1±0.5)×105,(0.8±0.5)×106;纯度分别为75％～ 85％,91％～95％,91％～95％；活率均高于90％.结论用Nycodenz作为分离介质分离肝星状细胞的方案最优.
목적 비교불동적분리개질대소서원대간성상세포(hepatic stellate cells,HSCs)분리순화효솔적차이.방법 간문정맥원위관주,예관주불함개미리자적D-Hanks액급함유Ⅳ형효원매적관주액,취소화후적간장,분별채용Ficoll-Paque PLUS、Percoll、Nycodenz작위분층개질,밀도제도리심순화원대HSCs.현미경하관찰세포순도병계산세포득솔.추충람염색판단세포활솔.격광공취초현미경관찰원대HSCs적고유형광정황.유홍O염색관찰획득원대HSCs적순도정황.결과 채용Ficoll-Paque PLUS、Percoll、Nycodenz분리세포득솔분별위(1±0.5)×106,(1±0.5)×105,(0.8±0.5)×106;순도분별위75％～ 85％,91％～95％,91％～95％；활솔균고우90％.결론 용Nycodenz작위분리개질분리간성상세포적방안최우.
Objective To compare the advantages and disadvantages of the three isolation regents for isolating primary hepatic stellate cells(HSCs) of mice.Methods The mice HSCs was isolated by in situ perfusion of per-perfusion solution,perfusion with collagenase Ⅳ.After digestion,Ficoll-Paque PLUS,perco11 and Nycodenz were use for the gradient centrifugation.To calculate the quantity,purity and the viability of the HSCs.The autofluorescence and Oil O staining were used to identify primary HSCs.Results The quantity of HSCs was (1.0 ± 0.5) × 106,(1.0 ± 0.5) × 105,(0.8 ± 0.5) × 106/ g_liver,respectively.Using Ficoll-Paque PLUS,percoll and Nycodenz,the purity of primary HSCs were 75％-85％,91％-95％,91％-95％,respectively.The viability were all above 90％.Conclusion compared with Ficoll-Paque PLUS and percoll,the Nycodenz is the the best separating medium for isolation and purification of mice primary HSCs.