|本期目录/Table of Contents|

[1]颜彩玲,张慧,周瑞祥.流式细胞术检测胞内细胞因子的经验分析[J].福建医科大学学报,2017,51(05):303-310.
 YAN Cailing,ZHANG Hui,ZHOU Ruixiang.The Empirical Analysis of Intracellular Cytokines Detection with Flow Cytometry[J].Journal of Fujian Medical University,2017,51(05):303-310.
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流式细胞术检测胞内细胞因子的经验分析(PDF)
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《福建医科大学学报》[ISSN:1672-4194/CN:35-1192/R]

卷:
第51卷
期数:
2017年05期
页码:
303-310
栏目:
论著
出版日期:
2017-10-30

文章信息/Info

Title:
The Empirical Analysis of Intracellular Cytokines Detection with Flow Cytometry
文章编号:
16724194(2017)05030308
作者:
颜彩玲1 张慧2 周瑞祥1
作者单位: 福建医科大学,福州3501221.基础医学院;2. 新药安全性评价中心
Author(s):
YAN Cailing1 ZHANG Hui2 ZHOU Ruixiang1
1. School of Basic Medical Sciences,Fujian Medical University,Fuzhou 350122,China; 2. Fujian Center for Safety Evaluation of New Drug,Fujian Medical University,Fuzhou 350122,China
关键词:
流式细胞术 细胞因子类 小鼠
Keywords:
KEY WORDS:flow cytometrycytokinesmice
分类号:
R332; R341.1; R392.21; R977.6
DOI:
-
文献标志码:
A
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。
Abstract:
ABSTRACT:ObjectiveTo investigate factors that may affect the detection of intracellular cytokines with flow cytometry.MethodsSplenocytes 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α after fixation and permeabilization.Controls were designed for both experiments.ResultsThe 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α producing cells between the fresh and frozen samples(P>0.05).Expression of TNFα in cells stimulated by PMA/Ionomycin, antiCD3/CD28, or combined stimulation groups were significantly higher than controls(P<0.05).ConclusionMany 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.

参考文献/References:

[1]袁芳. Th1/Th2和Th17/Treg平衡失调与宫颈癌发生发展关系及术后CD4+淋巴细胞亚群的改变[D]. 济南:山东大学医学院妇产科,2014.
[2]李路远, 刘俊燕, 钟岗,等. 流式细胞术检测小鼠脾细胞内细胞因子刺激方案的筛选[J].中国病原生物学杂志,2010,5(7):485489.
[3]任兴昌,方黎,陈洪勋. 实体组织单细胞悬液制备方法[J].临床与实验病理学杂志, 2007,23(4):490491.
[4]Peng H, Sun R, Zhang Q,et al. Interleukin 33 mediates type 2 immunity and inflammation in the central nervous system of mice infected with angiostrongylus cantonensis[J].J Infectious Diseases,2012,207(5):860869.
[5]孙黎飞. 细胞免疫学实验研究方法[M].北京:人民军医出版社,2009:2324.
[6]陈朱波, 曹雪涛. 流式细胞术——原理·操作及应用[M].2版.北京:科学出版社,2014:5255.
[7]Sheng H, Wang Y,Jin Y,et al. A critical role of IFNgamma in priming MSCmediated suppression of T cell proliferation through upregulation of B7H1[J]. Cell Research,2008,18(8):846857.
[8]Krieg C, Han P, Stone R,et al. Functional analysis of B and T lymphocyte attenuator engagement on CD4+ and CD8+ T cells[J].J Immunology,2005,175(10):64206427.
[9]Li X, Murray F, Koide N,et al. Divergent requirement for Gαs and cAMP in the differentiation and inflammatory profile of distinct mouse Th subsets[J]. J Clinical Investigation,2012,122(3):963973.
[10]Zago C A, Bortoluci K R, Sardinha L R,et al. AntiIL2 treatment impairs the expansion of Treg cell population during acute malaria and enhances the Th1 cell response at the chronic disease[J]. PloS One,2012,7(1):264264.
[11]Amidzadeh Z, Behbahani A B, Erfani N,et al. Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry[J]. Avicenna J Med Biotechnol,2014,6(1):3846.

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备注/Memo

备注/Memo:
基金项目: 福建省科技计划项目(2012Y0033) 作者单位: 福建医科大学,福州3501221.基础医学院;2. 新药安全性评价中心 作者简介: 颜彩玲,女,实验师,发酵工程硕士 通讯作者: 周瑞祥. Email: 1096330863@qq.com
更新日期/Last Update: 2017-10-30