流式细胞仪分选小鼠Kupffer细胞方法的建立与评价

Establishment and evaluation of flow cytometry method for isolation of Kupffer cells from mouse

目的:建立流式细胞仪分选小鼠肝脏Kupffer细胞(KCs)的方法。方法:小鼠肝脏经胶原酶灌注后密度梯度离心法分离非实质细胞,流式细胞分选仪分选单细胞悬液中7-AAD-CD45^(+)CD11b^(+)F4/80^(+)肝脏KCs并检测分选后细胞纯度,台盼蓝染色检测细胞活力,荧光微球吞噬实验检测细胞吞噬功能,CD86、CD206染色鉴定KCs细胞分型,脂多糖(LPS)刺激后检测炎性因子TNF-α、IL-1β的表达情况。结果:经过密度梯度分离和流式细胞分选,获得的7-AAD-CD45^(+)CD11b^(+)F4/80^(+)细胞纯度为(96.1±1.8)%,细胞存活率(96.5±2.1)%,分选后CD86^(+)和CD206^(+)所占比例分别为(92.3±1.6)%、(2.1±0.8)%,吞噬荧光微球的细胞百分比为(84.7±3.6)%,LPS刺激8h后,培养上清液中TNF-α、IL-1β显著升高(P<0.001)。结论:流式细胞分选仪可快速高效分离小鼠肝脏KCs,分选后的KCs保持良好细胞活性和吞噬功能,LPS刺激可诱导其分泌大量炎性因子。

Mouse livers were perfused by collagenase and nonparenchymal cells were separated by density gradient centrifugation.Flow cytometry was used to separate 7-AAD-CD45^(+)CD11b^(+)F4/80^(+)KCs from single cell suspension and test the purity of cells after sorting,The survival rate of these cells was detected by trypan blue staining,fluorescent microspheres uptake test was used to detect the phagocytic activity.KCs subgroups were identified by CD206 and CD86 staining.The expression of inflammatory factors TNF-α、IL-1βwas detected after LPS stimulation.The purity of 7-AAD-CD45^(+)CD11b^(+)F4/80^(+)cells was(96.1±1.8)%,the survival rate was(96.5±2.1)%,the percentages of CD86^(+)and CD206^(+)were(92.3±1.6)%,(2.1±0.8)%,and the percentage of cells phagocytized fluorescent microspheres was(84.7±3.6)%.After LPS stimulation for 8h,TNF-αand IL-1βin the culture supernatant increased significantly(P<0.001).This study demonstrates that flow cytometry is highly efficient in isolation of KCs from mouse liver,the sorted KCs maintain good cell activity and phagocytic function,LPS stimulation can induce them to secrete a large number of inflammatory factors.