小鼠肺组织单个核细胞三种分离方法的比较研究

Comparative Study on Three Methods for Isolating Mononuclear Cells from Mouse Lung Tissue

该文综合比较了三种不同方法分离小鼠肺组织单个核细胞的效果,并提供了一种能够有效分离小鼠肺脏全免疫细胞亚群的方法。将15只6~8周龄雌性C57BL/6小鼠随机分为三组(n=5只/组),分别为研磨+淋巴细胞分离液梯度离心分离组(M1组)、胶原酶Ⅳ消化+淋巴细胞分离液梯度离心分离组(M2组)和胶原酶Ⅳ消化分离组(M3组)。采用颈椎脱臼处死法处死小鼠,摘取肺脏,再分别采用上述三种方法制备小鼠肺脏单个核细胞悬液,于显微镜下拍照并计数,流式细胞术检测单个核细胞悬液中不同免疫细胞亚群的比例和数量,采用SPSS 26.0软件进行统计分析。结果显示,M3组制备的肺单个核细胞数量显著高于M1和M2组(F=44.49,P<0.001);M2组制备的肺单个核细胞中NK(F=16.68,P<0.001)、CD4^(+)T(F=12.28,P<0.01)和B细胞(F=4.49,P<0.05)比例均显著高于M1和M3组;M3组制备的肺CD3^(+)T、MDSC、中性粒细胞和间质巨噬细胞的比例显著高于M1和M2组;M3组制备的肺NK、CD3^(+)T、CD4^(+)T、CD8^(+)T、B细胞以及髓系细胞(MDSC、中性粒细胞、DC、肺泡巨噬细胞和间质巨噬细胞)的数量均显著高于M1和M2组。应用胶原酶Ⅳ消化直接分离法制备的小鼠肺组织单个核细胞得率最高,可有效分离到小鼠肺脏NK、T、B等淋巴细胞以及髓系来源免疫细胞亚群,且操作过程简便。该研究为肺部相关疾病的免疫学发病机制研究提供实验基础。

This study comprehensively compared the effects of three different methods for isolating mononuclear cells from mouse lung tissue,and provided an effective method for isolating the subpopulations of mouse lung immune cells.Fifteen female C57BL/6 mice(6-8 weeks old)were randomly divided into three groups(n=5/group),including the grinding with lymphocyte separation solution gradient centrifugation group(M1 group),collagenase IV digestion with lymphocyte separation solution gradient centrifugation group(M2 group),and collagenase IV digestion group(M3 group).The mice were sacrificed by cervical dislocation,and their lungs were extracted.The suspension of lung mononuclear cells was prepared by the above three methods,and photographed and counted under the microscope.The proportion and number of different immune cell subsets in lung tissues were detected by flow cytometry.Statistical analysis was conducted using SPSS 26.0 software.The results showed that the number of lung mononuclear cells in the M3 group was significantly higher than that in the M1 and M2 groups(F=44.49,P<0.001).the proportion of NK(F=16.68,P<0.001),CD4^(+)T(F=12.28,P<0.01)and B cells(F=4.49,P<0.05)in lung mononuclear cells in M2 group were higher than that in the M1 and M3 groups,and the proportion of CD3^(+)T,MDSC,neutrophils and lung interstitial macrophages prepared in M3 group was significantly higher than that in the M1 and M2 groups;the number of NK cells,CD3^(+)T,CD4^(+)T,CD8^(+)T,B cells,and myeloid cells(MDSC,neutrophils,DC and macrophages)prepared in M3 group were higher than that in the M1 and M2 groups.M3 group obtained the highest yield of lung mononuclear cells in mouse,and this method could effectively separate the lung NK,T,B lymphocytes and subpopulations of myeloid derived immune cells.This study provides an experimental basis for the immunological pathogenesis of lung related diseases.