基于流式细胞术的组蛋白γ-H2AX磷酸化检测方法的建立

doi:10.3969/j.issn.1004-616x.2017.04.008

癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (4): 284-288.doi: 10.3969/j.issn.1004-616x.2017.04.008

基于流式细胞术的组蛋白γ-H2AX磷酸化检测方法的建立

黄鹏程, 周长慧, 李申宁, 常艳

中国医药工业研究总院, 国家上海新药安全评价研究中心, 上海 201203

收稿日期:2016-12-23 修回日期:2017-04-13 出版日期:2017-07-31 发布日期:2017-07-31
通讯作者: 常艳,E-mail:ychang@ncdser.com E-mail:ychang@ncdser.com
作者简介:黄鹏程,E-mail:pchuang@ncdser.com。
基金资助:
上海市科技人才项目基金（15XD1523900）；上海市研发公共服务平台专项（15DZ2290300）

Establishment of detection method for histone γ-H2AX phosphorylation based on flow cytometry

HUANG Pengcheng, ZHOU Changhui, LI Shenning, CHANG Yan

China State Institute of Pharmaceutical Industry, National Shanghai Center for New Drug Safety Evaluation & Research, Shanghai 201203, China

Received:2016-12-23 Revised:2017-04-13 Online:2017-07-31 Published:2017-07-31

摘要/Abstract

摘要：

目的：建立基于流式细胞术的生物标记物γ-H2AX自动化检测方法，并探讨此方法用于药物早期遗传毒性筛选和遗传毒性评价的前景。方法：试验分为不添加体外代谢活化系统（-S9）长时（24 h）处理组和添加体外代谢活化系统（+S9）短时（4 h）处理组，分别采用CCK-8方法选择4个不同浓度的依托泊苷（ETO）和环磷酰胺（CP）处理人成淋巴TK6细胞，24 h后收获细胞，采用连接荧光分子的γ-H2AX抗体和核酸染料SYTOX Green双色标记，使用高通量流式细胞仪分析组蛋白H2AX磷酸化（γ-H2AX）阳性的细胞比例。结果：与溶剂对照组比较，-S9 24 h处理组，不同浓度的依托泊苷诱导产生的γ-H2AX阳性细胞比例显著增加，量效关系明显（r=0.999，P < 0.05）；+S9 4 h处理组，不同浓度的环磷酰胺诱导产生的γ-H2AX阳性细胞比例亦显著增加，量效关系明显（r=0.988，P < 0.05）。结论：流式细胞仪可快速、准确地分析体外培养的TK6细胞中γ-H2AX的变化，本试验初步建立了基于流式细胞术的组蛋白γ-H2AX磷酸化检测方法。

关键词: TK6细胞, γ-H2AX, 流式细胞术, 组蛋白, 磷酸化

Abstract:

OBJECTIVE: To established a method for automated detection of flow cytometry-based biomarker γ-H2AX,and to discussed the prospect of this method in drug screening for early genotoxicity. METHODS: The experiment was divided into two groups:-S9 group (long time treatment for 24 hours) and +S9 group (short time treatment for 4 hours). Then,4 suitable concentrations of Cyclophosphamide and Etoposide were selected using the CCK-8 assay. Treated TK6 cells were harvested according to standard operation procedures. After harvest,cells were double labeled with nucleic acid SYTOX Green and Alexa Fluor 647 conjugated anti-γ-H2AX antibody. BD Accuri C6 Flow cytometry was used to analyze the percentage of phosphorylated histone H2AX (γ-H2AX) cells. RESULTS: In the -S9 long-time treatment group,the ratio of γ-H2AX positive cells increased significantly compared with the solvent control group in different concentrations of Etoposide,and it showed a significant dose-dependent relationship. In the +S9 short-time treatment group,a significant increase in different concentrations of Cyclophosphamide was observed. CONCLUSION: Flow cytometry rapidly and accurately detected changes of γ-H2AX in cultured TK6 cells in vitro. Therefore,it is possible to detect γ-H2AX based on flow cytometry.

Key words: TK6 cell, γ-H2AX, flow cytometry, histone, phosphorylation

中图分类号:

Q355

黄鹏程, 周长慧, 李申宁, 常艳. 基于流式细胞术的组蛋白γ-H2AX磷酸化检测方法的建立[J]. 癌变·畸变·突变, 2017, 29(4): 284-288.

HUANG Pengcheng, ZHOU Changhui, LI Shenning, CHANG Yan. Establishment of detection method for histone γ-H2AX phosphorylation based on flow cytometry[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(4): 284-288.

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基于流式细胞术的组蛋白γ-H2AX磷酸化检测方法的建立

Establishment of detection method for histone γ-H2AX phosphorylation based on flow cytometry

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