医学分子生物学杂志2011，Vol.8Issue(6)：484-488,5.DOI:10.3870/j.issn.1672-8009.2011.06.004
小鼠Kupffer细胞分离及细胞培养
Isolation of Mouse Kupffer Cells and Their Primary Culture
摘要
Abstract
Objective Kupffer cells ( KCs ) were isolated by perfusion of collagenase in vivo, discontinuous density gradient centrifugation, and selective adherence. The yield and purity of KCs were analyzed in different group to investigate the best isolated method of KCs. Methods Mouse KCs were isolated by collagenase digestion and the method were randomly divided into 4 groups according to the specific steps: without collagenase in situ perfusion and gradient centrifugation of three layer group ( A group ), without collagenase in situ perfusion and gradient centrifugation of double layer group ( B group ), collagenase perfusion in situ and gradient centrifugation of three layer group ( C group ) and collagenase perfusion in situ and gradient centrifugation of double layer group ( D group ) . With F4/80 ( BM8 ) and swallowing ink immunostaining experiments to determine cell purity and function, using trypan blue dye test to determine cell viability, the survival time of KCs, morphological changes, as well as the effect of different methods were observed. Results KCs isolated freshly were just quasi-circular, and 1 h after inoculation cells were harvested at this time of high purity, but with relatively low cell yield. Four hours after inoculation KCs were harvested with relatively high yield. The cells were cultured for 28 d still alive. Trypan blue staining showed about 90% of cell vitality in each group. Immunohistochemistry and immunofluores-cence showed that cells isolated were KCs. Conclusion Collagenase perfusion in vivo and gradient can increase the yield of KCs and gradient centrifugation of double layer can increase the purity of I-solated KCs. In vivo perfusion of collagenase is important to improve the yield of KCs and separationof double layer can improve the isolated effect The method for isolation and culture of Kupffer cells is convenient, efficient, stable and cultured Kupffer cells retain naive biological characteristics.关键词
Kupffer细胞/细胞分离/梯度离心法/细胞原代培养Key words
Kupffer cells/ cell isolation/ density gradient centrifugation/ cell primary culture / mouse分类
医药卫生引用本文复制引用
朱荣涛,魏思东,李培志,龚建平..小鼠Kupffer细胞分离及细胞培养[J].医学分子生物学杂志,2011,8(6):484-488,5.基金项目
国家自然科学基金(No.81070374) （No.81070374）
