中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2014年
1期
64-66
,共3页
李红敏%任慧%郝晓花%黄玉波%魏红山
李紅敏%任慧%郝曉花%黃玉波%魏紅山
리홍민%임혜%학효화%황옥파%위홍산
肝硬化%细胞,培养的%细胞/分离和提纯%小鼠
肝硬化%細胞,培養的%細胞/分離和提純%小鼠
간경화%세포,배양적%세포/분리화제순%소서
Liver cirrhosis%Cells,cultured%Cells/isolation and purification%Mice
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.
目的 比較不同的分離介質對小鼠原代肝星狀細胞(hepatic stellate cells,HSCs)分離純化效率的差異.方法 肝門靜脈原位灌註,預灌註不含鈣鎂離子的D-Hanks液及含有Ⅳ型膠原酶的灌註液,取消化後的肝髒,分彆採用Ficoll-Paque PLUS、Percoll、Nycodenz作為分層介質,密度梯度離心純化原代HSCs.顯微鏡下觀察細胞純度併計算細胞得率.錐蟲藍染色判斷細胞活率.激光共聚焦顯微鏡觀察原代HSCs的固有熒光情況.油紅O染色觀察穫得原代HSCs的純度情況.結果 採用Ficoll-Paque PLUS、Percoll、Nycodenz分離細胞得率分彆為(1±0.5)×106,(1±0.5)×105,(0.8±0.5)×106;純度分彆為75% ~ 85%,91% ~95%,91% ~95%;活率均高于90%.結論 用Nycodenz作為分離介質分離肝星狀細胞的方案最優.
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.