采用基于γ-H2AX焦点分析的流式细胞术检测纳米金颗粒的遗传毒性  

Genetic toxicity of gold nanoparticles using flow cytometry based onγ-H2AX focus analysis

作　　者：姜睿琪 邓思思 王平 陈润桦 陈嘉琪 郑霖 冼静雯 黄泽愉 巩鑫超 刘楚珩 王晓炜 秦美蓉 JIANG Ruiqi;DENG Sisi;WANG Ping;CHEN Runhua;CHEN Jiaqi;ZHENG Lin;XIAN Jingwen;HUANG Zeyu;GONG Xinchao;LIU Chuheng;WANG Xiaowei;QIN Meirong(Shenzhen Institute for Drug Control,Key Laboratory for Monitoring and Evaluation of Cosmetics of National Medical Products Administration,Shenzhen 518057,Guangdong,China)

机构地区：[1]深圳市药品检验研究院,国家药品监督管理局化妆品监测评价重点实验室,广东深圳518057

出　　处：《癌变．畸变．突变》2024年第5期405-411,共7页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：广东省药品监督管理局2021年科技创新项目(2021TDB07)。

摘　　要：目的:建立基于磷酸化组蛋白H2AX(γ-H2AX)生物标志物的流式细胞术遗传毒性检测方法,为纳米金颗粒的遗传毒性评价提供参考。方法:在加与不加体外代谢活化系统(S9)的条件下,分别设+S9短时处理组,选择终浓度为7.5、3.75和1.875μg/mL的环磷酰胺(CP)处理4 h;-S9长时处理组,选择终浓度为6.4、1.6和0.4μg/mL的依托泊苷(ETO)连续接触24 h,建立γ-H2AX生物标志物的流式细胞术检测方法。选择两种纳米金颗粒(样品1为溴化十六烷基三甲基溴化铵修饰,样品2为聚乙二醇修饰)作为实验材料,根据细胞毒性试验结果样品1和2分别选取3个不同浓度处理人成淋巴TK6细胞,分为+S9短时4 h组和-S9长时24 h组处理后收获细胞,采用γ-H2AX荧光抗体进行标记,流式细胞术分析γ-H2AX阳性细胞比例。同时设CP或ETO阳性对照和相应溶剂对照组。结果:与溶剂对照组相比,在有或无S9条件下,阳性对照组诱导产生的γ-H2AX阳性细胞比例显著增加(P<0.05),并存在剂量反应关系(CP:R2=0.837,P=0.01;ETO:R2=0.903,P<0.01),说明方法成功建立。在加与不加S9的条件下,两种纳米金样品各剂量组的γ-H2AX阳性细胞比例较溶剂对照组差异均无统计学意义(均为P>0.05)。结论:本实验建立了基于γ-H2AX焦点分析的流式细胞术遗传毒性检测方法,可用于纳米金颗粒的遗传毒性评价。OBJECTIVE:To use a flow cytometry genotoxicity assay based onγ-H2AX biomarker for improvement of genotoxicity evaluation of gold nanoparticles.METHODS:Final concentrations of 7.5,3.75,and 1.875μg/mL of Cyclophosphamide were used as the positive controls for establishment of theγ-H2AX assay in+S9 group(4 hours of short treatment)and final concentrations of 6.4,1.6,and 0.4μg/mL of Etoposide were used as the positive controls for the establishment of theγ-H2AX assay in-S9 group(24 hours of long treatment)to establish a flow cytometry genotoxicity assay based onγ-H2AX biomarker.Two kinds of modified gold nanoparticles(sample 1 modified with cetyltrimethylammonium bromide and sample 2 modified with polyethylene glycol)were selected as experimental materials,which was diluted into three concentrations based on cytotoxicity test.The treated TK6 cells were then harvested and labeled with anti-H2AX fluorescent antibody,and Flow cytometry was used to analyze the percentage ofγ-H2AX,which has been shown to be positively related to the extent of the genotoxicity.RESULTS:Both groups with and without S9 showed significant increase in the percentage ofγ-H2AX cells compared with the solvent controls(P<0.05)and these increasements were dose-related(CP:R2=0.837,P=0.01;ETO:R2=0.903,P<0.01),thus validating the reliability of the method.On the other hand,the two gold nanoparticles products did not show significant differences in the ratio ofγ-H2AX cells(P>0.05).CONCLUSION:Flow cytometry was useful in accurately detecting changes ofγ-H2AX in cultured TK6 cells.This assay can be an approach for indicating the genotoxicity of toxic substances.

关 键 词：Γ-H2AX 纳米金 流式细胞术 TK6细胞 

分 类 号：R394.6[医药卫生—医学遗传学]