Percoll密度梯度离心法分离中枢神经系统组织中的单个核细胞
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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.
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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[2]Ferretti M T,Merlini M,Spani C,et al.T-cell brain infiltration and immature antigen-presenting cells in transgenic models of Alzheimer's disease-like cerebral amyloidosis[J].Brain Behav Immun,2016,54:211-25.
[3]Dittel B N.CD4 T cells:Balancing the coming and going of autoimmune-mediated inflammation in the CNS[J].Brain Behav Immun,2008,22(4):421-30.
[4]郑明华,唐玉兰,韦俊杰,等.载脂蛋白E拟肽对实验性自身免疫性脑脊髓炎小鼠脑脊髓CD4+、CD8+T淋巴细胞表达的影响[J].临床神经病学杂志,2013,3:198-201.[4]Zheng M H,Tang Y L,Wei J J,et al.Effect of apolipoprotein Emimetic peptides on expressions of CD4+,CD8+T cell of brain,spinal cord in mice with experimental autoimmune encephalomyelitis[J].J Clin Neurol,2013,3:198-201.
[5]龚业莉,李佳楠,黄丽霞.高丽参提取液对大鼠自身免疫性脑脊髓膜炎的保护作用[J].中国免疫学杂志,2016,32:1632-5.[5]Gong Y L,Li J N,Huang L X.Protective effect of korean red ginseng extract in experimental autoimmune en-cephalomyelitis rats[J].Chin J Immunol,2016,32:1632-5.
[6]王赵伟,厉芳,吴承龙,等.沙利度胺抑制实验性自身免疫性脑脊髓炎与促进Treg细胞中枢募集有关[J].浙江医学,2017,22:1957-60,1980.[6]Wang Z W,Li F,Wu C L,et al.Thalidomide alleviates experimental autoimmune encephalomyelitis through promoting Treg cell recruitment in rats[J].Zhejiang Med,2017,22:1957-60,1980.
[7]Pino P A,Cardona A E.Isolation of brain and spinal cord mononuclear cells using percoll gradients[J].J Vis Exp,2011,48(48):e2348.
[8]Garcia J A,Cardona S M,Cardona A E.Isolation and analysis of mouse microglial cells[J].Curr Protoc Immunol,2014,104:unit14.14.35.doi:10.1002/0471142735.im1435s104.
[9]Miller S D,Karpus W J.Davidson T S.Experimental autoimmune encephalomyelitis in the mouse[M].Curr Protoc Immunol,2010,88(1):15.1.1-20.
[10]Jiang S,Dong C.A complex issue on CD4(+)T-cell subsets[J].Immunol Rev,2013,252(1):5-11.
[11]刘端勇,黄敏芳,徐荣,等.黄芪多糖对结肠炎大鼠小肠PP结中T淋巴细胞亚群的调节作用[J].中国药理学通报,2015,31(9):1328-9.[11]Liu D Y,Huang M F,Xu R,et al.Effect of Astragalus polysaccharides in regulating the T lymphocyte subgroup in intestinal PPs of rat with colitis[J].Chin Pharmacol Bull,2015,31(9):1328-9.
[12]Zhao D,Xu A,Dai Z,et al.WNT5A transforms intestinal CD8alphaalpha(+)IELs into an unconventional phenotype with pro-inflammatory features[J].BMC Gastroenterol,2015,15:173.
[13]Kristian T.Isolation of mitochondria from the CNS[M].Curr Protoc Neurosci,2010,Chapter 7:Unit 7 22.doi:10.1002/0471142301.ns0722S52.