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

doi:10.3969/j.issn.1004-616x.2024.05.012

摘要/Abstract

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1.875 μg/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0.4 μg/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。

关键词: γ-H2AX, 纳米金, 流式细胞术, TK6细胞

Abstract: OBJECTIVE： To use a flow cytometry genotoxicity assay based on γ-H2AX biomarker for improvement of genotoxicity evaluation of gold nanoparticles. METHODS： Final concentrations of 7.5，3.75，and 1.875 μg/mL of Cyclophosphamide were used as the positive controls for establishment of the γ-H2AX assay in +S₉ group (4 hours of short treatment) and final concentrations of 6.4，1.6，and 0.4 μg/mL of Etoposide were used as the positive controls for the establishment of the γ-H2AX assay in -S₉ group (24 hours of long treatment) to establish a flow cytometry genotoxicity assay based on γ-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 of γ-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 of γ-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 of γ-H2AX cells (P>0.05). CONCLUSION： Flow cytometry was useful in accurately detecting changes of γ-H2AX in cultured TK6 cells. This assay can be an approach for indicating the genotoxicity of toxic substances.

Key words: γ-H2AX, gold nanoparticles, flow cytometry, TK6 cell

中图分类号:

R394.6

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. 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[J]. 癌变·畸变·突变, 2024, 36(5): 405-411.

JIANG Ruiqi, DENG Sisi, WANG Ping, CHEN Runhua, CHEN Jiaqi, ZHENG Lin, XIAN Jingwen, HUANG Zeyu, GONG Xinchao, LIU Chuheng, WANG Xiaowei, QIN Meirong. Genetic toxicity of gold nanoparticles using flow cytometry based on γ-H2AX focus analysis[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2024, 36(5): 405-411.

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