采用流式细胞术分选EAE模型小鼠小胶质细胞  

Isolation of microglia from EAE mice by flow cytometry

作　　者：浦颖艳[1] 孙定亚 黄爱军[1] 曹莉[1] 

机构地区：[1]第二军医大学神经生物学教研室教育部分子神经生物学重点实验室,上海200433

出　　处：《中国医药生物技术》2016年第4期295-299,共5页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金面上项目(81371326)

摘　　要：目的获取实验性自身免疫性脑脊髓炎(EAE)模型小鼠小胶质细胞,并检测其特性。方法取8周龄,雌性C57BL/6小鼠行EAE造模,待至发病高峰期(第15天)时以PBS灌注后取小鼠后脑和脊髓,切碎后加胰酶消化,消化完成后通过100μm滤网滤过,而后采用Percoll密度梯度离心法分离获取单个核细胞,进一步利用CD11b与CD45抗体染色,通过流式细胞仪分选CD11b^+CD45^(high)和CD11b^+CD45^(low)细胞,即分别得到相对活化和静息的小胶质细胞,最后对流式分选获取的小胶质细胞行qPCR检测。结果通过形态比较,获得了高纯度的小胶质细胞。FIZZ-1基因行qPCR检测,结果显示在EAE急性期M2型小胶质细胞明显增多,符合文献报道。结论该方法能从EAE小鼠体内分离获取高纯度的小胶质细胞,可用于相关目的基因后续检测。Objective To obtain microglia from EAE mice for q PCR analysis. Methods Firstly, after the induction of EAE model using C57BL/6 mice at the age of 8 week, EAE or control mice were perfused and the spinal cords and tritocerebrums were isolated. Then the mononuclear cells were collected by density gradient centrifugation after digestion. Secondly, microglia were separated by flow cytometry sorting after anti-CD11 b and anti-CD45 staining. Finally, collected microglia were analyzed by qPCR. Results Highly purified microglia were obtained observed by the cell morphology. The following qPCR analysis showed a high expression of FIZZ-1 which is a M2 marker in EAE mice, and this was consistent with previous report. Conclusion Highly purified microglia could be obtained by our method from EAE mice and the cells were suitable for following gene expression analysis.

关 键 词：小胶质细胞 流式细胞术 实验性自身免疫性脑脊髓炎 

分 类 号：R-332[医药卫生] R744.5