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

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。

bOBJECTIVE：To established a method for automated detection of flow cytometry-based biomarkerγ-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 ofγ-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 ofγ-H2AX in cultured TK6 cellsin vitro. Therefore,it is possible to detectγ-H2AX based on flow cytometry.