大鼠Kupffer细胞分离方法的优化

The optimization of rat Kupffer cell insolation method

目的:对大鼠Kupffer细胞分离方法进行优化。方法:采用D-Hanks液和Ⅳ型胶原酶液两步原位灌注法结合离体消化,差速离心法和Percoll分离液密度梯度离心法分离并选择性贴壁纯化Kupffer细胞。采用328 nm波长自发荧光法、CD68和CD163免疫荧光染色法及吞噬实验鉴定Kupffer细胞。结果:Kupffer细胞获得量为9.8×10~6个/鼠肝,其中活细胞率占93%以上,证实细胞为Kupffer细胞且吞噬功能正常。结论:该方法花费时间短,相对简便、经济,且细胞数量、功能活性和纯度等均达到实验要求。

Objective：To optimize the insolation method of rat kupffer cells. Method：Kupffer cells were isolated by in situ perfusion of D-Hanks solution and type Ⅳ collagenase solution combined with in vitro digestion,differential centrifugation and density gradient centrifugation of Percoll,and purified by selective attachment. Kupffer cells were identified by 328 nm wave length autofluorescence,CD68 and CD163 immunofluorescence staining and phagocytosis test. Results：Kupffer cells were obtained 9.8×10~6 cells per rat liver,among which the percentage of living cells was over 93%. It was confirmed that the cells were Kupffer cells and their phagocytic function was normal. Conclusion：This method takes a short time,relatively simple,economical,and the number of cells,functional activity and purity,which is up to the experiment requirements.