小鼠肝枯否细胞和肝星状细胞分离提取方法的比较及优化  

Comparison and optimization of isolation and extraction methods for Kupffer and hepatic stellate cells from mouse liver

作　　者：马文萌 吴昊[1] 闫昱彤 孙逊 郑倩倩 MA Wenmeng#;WU Hao#;YAN Yutong;SUN Xun;ZHENG Qianqian(China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学,沈阳110122

出　　处：《中国比较医学杂志》2023年第12期68-74,共7页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金青年基金(32200977);辽宁省自然科学基金(2022-MS-237);辽宁省教育厅科研项目(LJKMZ20221204)。

摘　　要：目的 探讨小鼠肝枯否细胞和肝星状细胞的提取及纯化方法,为小鼠原代肝非实质细胞分离提取方法学提供参考和建议。方法 以体内胶原酶灌注消化为基础,使用Percoll、OptiPrep等不同的试剂和浓度梯度分别提取C57BL/6小鼠的肝枯否细胞和肝星状细胞,并通过流式细胞仪、免疫荧光等方法验证纯化细胞的纯度。结果 两层Percoll法提取枯否细胞和两层OptiPrep法提取肝星状细胞可行,纯度可达90%以上,细胞得率为1~2×10~7/肝,细胞存活率在90%以上。细胞原代培养48 h后,枯否细胞F4/80阳性细胞、肝星状细胞α-SMA阳性细胞均达90%以上。结论 小鼠肝中枯否细胞和肝星状细胞的分离提取方法完善可靠且具有成本效益和可重复性。Objective To explore the extraction and purification method of Kupffer and hepatic stellate cells from mouse liver and provide references and suggestions for the separation and extraction method ology of primary non-parenchymal cells from mouse liver.Methods After in vivo collagenase perfusion digestion,various reagents and method,such as Percoll and OptiPrep,were used to extract C57BL/6 mice Kupffer and hepatic stellate cells,and evaluate their purity by flow cytometry and immunofluorescence.Results The two-layer Percoll method to extract Kupffer cells and the two-layer OptiPrep method to extract hepatic stellate cells were feasible,and purity reached>90%.The cell yield was 1~2×107/liver,and the cell survival rate was>90%.After 48 hours of primary cell culture,the number of F4/80-positive Kupffer cells andα-SMA-positive hepatic stellate cells reached>90%.Conclusions The separation and extraction method of Kupffer and hepatic stellate cells from mouse liver are perfect,reliable,cost-effective,and reproducible.

关 键 词：枯否细胞 肝星状细胞 灌注 原代细胞 纯度 

分 类 号：R-33[医药卫生]