小鼠肝脏中枯否细胞和肝星状细胞的分离提取
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中国医科大学

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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)。


Isolation and extraction of Kupffer cells and hepatic stellate cells in mouse liver
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China Medical University

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The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)

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    摘要:

    【】 目的 探讨小鼠肝脏枯否细胞和肝星状细胞的提取及纯化方法,为小鼠原代肝脏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小鼠的肝脏枯否细胞和肝星状细胞并通过流式细胞仪及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1-2×107/肝,细胞存活率在90%以上。细胞原代培养48h后,枯否细胞F4/80阳性细胞、肝星状细胞α-SMA阳性细胞均达75%以上。结论 小鼠肝脏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。

    Abstract:

    【】Objective It is to explore the extraction and purification methods of Kupffer cells and hepatic stellate cells from mouse liver, and provide reference and suggestions for the separation and extraction methodology of primary non parenchymal cells from mouse liver.Methods Based on in vivo collagenase perfusion digestion, different reagents and methods such as Percoll and OptiPrep were used to extract C57BL/6 mice Kupffer cells and hepatic stellate cells and evaluate the purity through flow cytometry and immunofluorescence methods. Results The two-layer Percoll method for extracting Kupffer cells and the two-layer OptiPrep method for extracting hepatic stellate cells are feasible,and the purity can reach more than 90%.The cell yield was 1~2×107/liver, the cell survival rate was more than 90%.After 48 hours of primary cell culture, the number of Kupffer cells F4/80-positive cells and hepatic stellate cells α-SMA-positive cells reached more than 75%. Conclusions The separation and extraction method of Kupffer cells and hepatic stellate cells from mouse liver is perfect, reliable, cost-effective and reproducible.

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  • 收稿日期:2023-08-30
  • 最后修改日期:2023-11-09
  • 录用日期:2023-12-04
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