采用流式细胞术分选EAE模型小鼠小胶质细胞

发布时间：2017-12-26 21:40

本文关键词：采用流式细胞术分选EAE模型小鼠小胶质细胞　出处：《中国医药生物技术》2016年04期 　论文类型：期刊论文

【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。
[Abstract]:Objective to obtain microglia in experimental autoimmune encephalomyelitis (EAE) model in mice and to detect its characteristics. Methods 8 week old female C57BL/6 mice EAE model, to the peak incidence (fifteenth days) with PBS perfusion after cerebral and spinal mouse, chopped after trypsin digestion, the digestion is completed by 100 m filter filtration, and then using Percoll density gradient centrifugation to obtain mononuclear cells, further use CD11b and CD45 antibody staining by flow cytometer (high) and CD11b~+CD45~ CD11b~+CD45~ (low) cells, which are relatively activated and resting microglia and microglia for qPCR. Finally get convection type sorting test. Results the microglia with high purity were obtained by comparison of the morphology. The FIZZ-1 gene was detected by qPCR. The results showed that the number of M2 microglia increased significantly in the acute phase of EAE, which was reported in the literature. Conclusion this method can be isolated from EAE mice to obtain high purity microglia and can be used for subsequent detection of related target genes.
【作者单位】：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;
【基金】：国家自然科学基金面上项目(81371326)
【分类号】：R-332;R744.5
【正文快照】： 小胶质细胞在中枢神经系统中约占12%[1],是中枢主要的固有免疫细胞。静息状态下的小胶质细胞主要发挥中枢神经系统的监视作用。中枢的病理损伤可以激活小胶质细胞,发生形态学和分子表达谱的变化,参与吞噬、抗原呈递、调节血脑屏障和调控炎症反应强度等多种作用。作为中枢神经

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