摘要
目的建立基于流式细胞术的大鼠外周血网织红细胞微核试验,检测阳性诱变剂甲基磺酸乙酯(ethyl methanesulphonate,EMS)和环磷酰胺(cyclophosphamide,CP)在所建立的试验方法中的表现。方法使用CD71-异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记大鼠外周血网织红细胞,核酸染料DRAQ5标记DNA,建立流式细胞术检测方案。采用该方案测定EMS和CP的遗传毒性时,将40只雄性SD大鼠随机分为8组,EMS染毒剂量为0、50、100、200mg/kg.bw,CP为0、5、10、15 mg/kg.bw,间隔24 h2次经口染毒。在给药前1 d和给药后24、48、72、96、120 h取尾静脉血,使用流式细胞术测定外周血网织红细胞和成熟红细胞微核率。结果本试验方法所得历史阴性对照成熟红细胞和网织红细胞背景微核率均数分别为0.04‰和0.42‰,标准差分别为0.02‰和0.18‰。在所建立的试验系统中观察到EMS及CP可诱导外周血网织红细胞微核显著升高。结论本实验室建立的基于CD71-FITC和DRAQ5双染的外周血流式微核技术操作简便快捷,背景微核率较低,检测结果稳定,在化学物的遗传毒性评价方面具有较大的推广价值和良好的应用前景。
Objective The aim of this work was to develop a study protocol for rat micronucleus test by flow cytometry, which was conducted on peripheral blood reticulocytes treated with known positive chemicals, ethyl methanesulphonate(EMS) and cyclophosphamide(CP). Methods CD71-fluorescein isothiocyanate(FITC) and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, so as to detect micronuclei in rat peripheral blood erythrocytes. In this experiment, 40 male SD rats were randomized divided to 8 groups, i.e. 3 treatment groups of EMS(0, 50, 100, 200mg/kg·bw), 3 treatment groups of CP(0, 5, 10, 15 mg/kg·bw) and 2 solvent control groups(distilled water). Tail vein blood specimens for micronucleus test were collected on-1 d(one day before gavage) and 24, 48, 72, 96, and 120 h. The numbers of micronucleus, red blood cells and reticulocytes were acquired by flow cytometer. Results For negative historical control data(total: 125), the mean percentages of micronucleated red blood cells and micronucleated reticulocytes were0.04‰ and 0.42‰, respectively, and the standard deviations were 0.04‰ and 0.42‰ respectively. The flow cytometric method consistently demonstrated highly sensitive responses for MN production at all concentrations and all time points of EMS and CP. Conclusion These data suggested that the method reported here, combined with easier operation, lower background and better precision, could be an efficient system in chemical safety assessment of genotoxicity.
作者
刘运杰
霍娇
岳茜岚
朱雪娇
曾珠
彭子豪
陈锦瑶
张立实
LIU Yun-jie;HUO Jiao;YUE Qian-lan;ZHU Xue-jiao;ZENG Zhu;PENG Zi-hao;CHEN Jin-yao;ZHANG Li-shi(West China School of Public Health,Food Safety Monitoring,Risk Assessment Key Laboratory of Sichuan,Chengdu Sichuan 610041,China)
出处
《现代预防医学》
CAS
北大核心
2019年第2期322-326,352,共6页
Modern Preventive Medicine
基金
国家自然科学基金面上项目(项目编号:8157120809)
关键词
微核试验
流式细胞术
遗传毒性
环磷酰胺
甲基磺酸乙酯
Micronucleus test
Flow cytometry
Genotoxicity
Cyclophosphamide
Ethyl methylsulfonate
作者简介
刘运杰(1992-),男,硕士研究生,研究方向:遗传毒性;通讯作者:张立实,E-mail:lishizhang_56@163.com.
