流式细胞术检测小鼠肝脏中嗜酸性粒细胞方法的建立

摘要

目的：建立稳定的流式细胞术检测小鼠肝脏组织浸润嗜酸性粒细胞的方法。方法华支睾吸虫感染4周小鼠及正常小鼠肝脏原位灌注后，制备肝组织单细胞悬液，Procell密度梯度离心获得肝脏白细胞；采用多色流式细胞术，前向角散射（ FSC）、侧向角散射（ SSC）及CD45等参数圈门，利用抗Gr－1、Siglec－F等分子的流式抗体对其中的嗜酸性粒细胞进行标记、检测分析。结果正常肝脏白细胞流式检测显示在前散射中等位置（ FSCmid ）、侧散较高处（ SSChig ）有一群细胞，表型多为CD45＋CD3－CD19－；华支睾吸虫感染后该群细胞比例明显增高，该群细胞不表达MHCⅡ类分子（MHCⅡ－），低表达Gr1（Gr1low），表达Siglec F（Siglec F＋）。结论流式细胞术检测CD45＋FSCmid SSChig Gr1low Siglec F＋细胞是一种稳定的检测及分析肝脏嗜酸性粒细胞数量及功能的方法。%Objective To establish a stable method for detection of the eosinophils infiltrating in the liver of mice by flow cytometry.Methods Normal mice and those infected with Clonorchis sinensis for four weeks underwent in situ per-fusion in the liver.Then, the monocyte suspension of the liver was prepared to isolate hepatic leukocytes.Then, the eo-sinophils were labeled using with anti-Gr-1, anti-SIgle-F and other flow cytometry antibodies.Results According to flow cytometry results, there was a group of cells in the middle former scattering ( FSC) and high side scatter ( SSC) in normal hepatic leukocytes, which was more obvious in Clonorchis sinensis infected mice.These cells expressed Siglec F (Siglec F+), with low expression of Gr1(Gr1low) and without expression of MHC Ⅱ molecules (MHCⅡ-).Conclu-sions Flow cytometry can be applied for stable detection of CD45 +FSCmidSSChigGr1lowSiglec F+cells so as to analyze the function of eosinophils in the liver.