| 蒋子剑,岳振生,杨毅聪,阮 柏,段娟丽,岳树强.小鼠肝血窦内皮细胞分离与鉴定新方法[J].,2018,(6):1034-1039 |
| 小鼠肝血窦内皮细胞分离与鉴定新方法 |
| Improvement Method of Isolation of Mouse Liver Sinusoidal Endothelial Cell |
| 投稿时间：2017-11-08 修订日期：2017-11-23 |
| DOI：10.13241/j.cnki.pmb.2018.06.007 |
| 中文关键词: 肝血窦内皮细胞 分离 鉴定 |
| 英文关键词: Liver sinusoidal endothelial cell Isolation Identification |
| 基金项目:国家自然科学基金面上项目（81370556） |
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| 中文摘要: |
| 摘要 目的：改进小鼠原代肝血窦内皮细胞的分离方法。方法：经过小鼠肝脏的原位灌洗、消化制备单细胞悬液、差速离心、密度梯度离心以及免疫磁珠分选等步骤，分离获得小鼠原代肝血窦内皮细胞，再通过流式细胞仪鉴定、细胞内吞功能染色以及对细胞超微结构的电子显微镜观察，对分离出的肝血窦内皮细胞进行鉴定。结果：肝血窦内皮细胞的平均得率为5.6 ×106个/只小鼠，细胞活性比率约为96 %左右；细胞流式鉴定结果显示新鲜分离出的肝血窦内皮细胞VEGFR3阳性率达到95.8 %，VEGFR2+CD31+双阳性细胞阳性率达到93.7 %。分选出的LSECs能够有效吞噬FITC-FSA和Dil-Ac-LDL。培养1天后肝血窦内皮细胞的微观结构，可见其特征性的窗孔和筛板。结论：本文总结的分离方法可以稳定、高效地获得小鼠原代肝血窦内皮细胞。 |
| 英文摘要: |
| ABSTRACT Objective: To improve the method of isolation of LSECs. Methods: The mouse primary liver sinusoidal endothelial cells were isolated by a series of methods, such as lavage of mouse liver in situ, digestion and making single cell suspension, differential centrifugation, density gradient centrifugation and magnetic activated cell sorting. Then the isolated liver sinusoidal endothelial cells were identified by flow cytometry, cell endocytosis staining and cell ultrastructure observation with electron microscopy. Results: The average yield and Viability of LSECs was respectively 5.6×106 per mouse and 96 %; the flow cytometry results showed that the VEGFR3 positive rate of isolated liver sinusoidal endothelial cells was 95.8 %, the VEGFR2+CD31+ double positive rate of LSECs was 93.7 %.The isolated LSECs could phagocytose FITC-FSA and Dil-Ac-LDL efficiently. The characteristic fenestrate and sieve plate of LSECs could be observed after 24 hours'cultivation in electron microscopy. Conclusion: The isolation method summarized in this article could steadily and efficiently obtain mouse primary LSECs. |
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