流式细胞术检测胞内细胞因子的经验分析  

The Empirical Analysis of Intracellular Cytokines Detection with Flow Cytometry

作　　者：颜彩玲[1] 张慧 周瑞祥[1] 

机构地区：[1]福建医科大学基础医学院,福州350122 [2]福建医科大学新药安全性评价中心,福州350122

出　　处：《福建医科大学学报》2017年第5期303-310,共8页Journal of Fujian Medical University

基　　金：福建省科技计划项目(2012Y0033)

摘　　要：目的探讨流式细胞术检测胞内细胞因子的注意事项。方法制备小鼠脾脏淋巴细胞悬液。采用细胞表面抗原CD4、CD25及细胞核内因子FOXP3染色检测调节T细胞,胞内因子TNF-α的检测采用PMA/Ionomycin,anti-CD3/CD28,PMA/Ionomycin与anti-CD3/CD28联合3种方案进行刺激,比较不同刺激条件下TNF-α胞内因子的表达情况,培养后各组细胞悬液采用固定破膜处理技术进行细胞表面抗原CD3、CD4及细胞内因子TNF-α染色。结果脾脏淋巴细胞悬液制备中研磨、裂解红细胞及染色过程中破膜等操作的不当,均导致前向/侧向(FSC/SSC)点图中目的细胞群模糊不清,从而影响后续分析。新鲜脾脏淋巴细胞得率、CD3^+T淋巴细胞得率及TNF-α表达率与冻存的脾脏各类细胞比较,差别无统计学意义(P>0.05)。胞内因子TNF-α检测结果显示,刺激组PMA/Ionomycin以及anti-CD3/CD28表达率显著高于未刺激组(P<0.05),PMA/Ionomycin与anti-CD3/CD28联合刺激组的表达率也显著高于未刺激组和独立刺激组(P<0.05)。结论流式细胞术胞内细胞因子的检测有很多影响因素,包括脾脏细胞悬液制备中的研磨、裂解红细胞操作,染色处理过程中的破膜、固定,上机检测过程中补偿的调节、对照的设置等均影响实验结果,检测前应注意摸索选择适合待测胞内因子的测定条件。Objective To investigate factors that may affect the detection of intracellular cytokines with flow cytometry. Methods Splenocytes from mice were disaggregated,and regulatory T cells（Treg）were detected by staining of CD4,CD25,and intranuclear transcription factor FOXP3. For the detection of TNF-α,splenocytes were stimulated with PMA/Ionomycin,anti-CD3/CD28 or PMA/Ionomycin plus anti-CD3/CD28,respectively. Following activation,the cells were stained with anti-CD3 APC and anti-CD4 FITC,then intracellularly stained to detect TNF-αafter fixation and permeabilization.Controls were designed for both experiments. Results The improper operations in the grinding,lysing erythrocytes,fixing and perming the cells all leaded to indistinct target cells of interest in the FSC/SSC dot plots,which affected the subsequent analysis. There were no significant differences in the percentages of lymphocytes,CD3^＋T lymphocytes,and TNF-αproducing cells between the fresh and frozen samples（P〈0.05）. Expression of TNF-αin cells stimulated by PMA/Ionomycin,anti-CD3/CD28,or combined stimulation groups were significantly higher than controls（P〈0.05）. Conclusion Many factors may affect the detection of intracellular cytokines by flow cytometry,including the spleen,lysing erythrocytes in the preparation of splenocytes suspension,fixing and perming the cells during dyeing,adjusting the compensation values,designing controlled trials and so on. Therefore,attentions should be paid to explore and select the suitable conditions for studying intracellular factors before testing.

关 键 词：流式细胞术 细胞因子类 小鼠 

分 类 号：R392[医药卫生—免疫学]