摘要
目的建立快速分离小鼠中枢神经系统(CNS)组织中单个核细胞的方法。方法以正常C57BL/6小鼠、实验性变态反应性脑脊髓炎(EAE)和阿尔茨海默病(AD)模型小鼠为研究对象,通过70%~30%Percoll密度梯度离心法,获取小鼠脑组织和脊髓中单个核细胞,利用台盼蓝染色检测细胞存活率后,通过流式细胞术进一步分析细胞的表型特点。结果分离得到的单个核细胞存活率在90%以上;流式细胞术分析结果表明,EAE和AD小鼠中浸润到CNS的淋巴细胞群比例明显升高,且介导炎症反应的Th1和Th17细胞亚群比例明显升高,而具有免疫调节作用的Treg细胞亚群比例明显下降。结论采用70%~30%Percoll密度梯度离心法从小鼠CNS中分离的单个核细胞存活率高,细胞活性不受分离过程的影响,可进一步利用流式细胞术分析细胞的表型特征。此方法操作简单快速,适用于对CNS淋巴细胞等单个核细胞的分析。
Aim To establish a rapid method to efficiently isolate mononuclear cells from central nervous system( CNS) tissues of mice that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.Methods For defining the feasibility and practicality of the method,wild-type C57BL/6 mice and two mouse models of CNS disease including EAE mice and APP/PS1 mice were used in this study.After the collection and homogenization of the brain and spinal cord tissues respectively,the mononuclear cells were isolated by spinning the 70%-30% Percoll gradients.Cell activities were detected by trypan blue staining,and the immune population that infiltrated CNS was identified by flow cytometry.Results The results of trypan blue staining showed that the survival rate of the isolated cells was above 90% in all groups.Flow cytometry analysis showed that the relative numbers of lymphocytes infiltrating CNS of EAE and AD mice increased significantly compared with wild-type C57BL/6 mice.In addition,the relative numbers of Th1 and Th17 cell subsets mediating the inflammatory response also increased significantly,while the decreased regulatory T cells frequency was observed in the two mouse models of CNS disease.Conclusions The cells isolated by the 70% ~ 30% Percoll gradients centrifugation can be effectively utilized for the identification of various immune cell populations in a single sample by flow cytometry.The method described in this article is simple and rapid in operation and with high survival rate and activity of the cells,which can be applied to the study of the mononuclear cells in CNS.
引文
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