基于三色流式细胞术的小鼠外周血微核试验方法的建立  

Three-color labeling method for flow cytometric measurement of micronucleus in mouse peripheral blood

作　　者：周长慧[1] 王征[1] 王庆利[2] 杨琛懋[1] 常艳[1] 

机构地区：[1]上海医药工业研究院国家上海新药安全评价研究中心,上海201203 [2]国家食品药品监督管理局药品审评中心,北京100038

出　　处：《癌变．畸变．突变》2011年第2期128-133,共6页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：上海科委专业技术服务能力建设基金资助项目(09DZ2290900)

摘　　要：目的:为克服传统显微镜检微核(micronucleus,MN)方法耗时、费力、主观等缺点,建立基于CD71、CD61荧光抗体和碘化丙啶(cyclophosphamidr,PI)的三色流式细胞术(flow cytometry,FCM)研究小鼠外周血微核的自动化检测方法。方法:经口单次给予小鼠0、20、40和80mg/kg环磷酰胺(CP),于不同时间点收集外周血;另一批小鼠给予CP40mg/(kg·d)经口灌胃,连续染毒5d,分别于给药前和每次给药后24h收集外周血。采用-80℃甲醇固定血细胞,CD71、CD61荧光抗体、RNA酶和PI孵育血细胞,以疟原虫感染的红细胞作为生物标准物调校FCM后,对CP诱导的小鼠外周血中含MN的CD71^+网织红细胞(reticulocytes,RET)进行检测。结果:单次染毒后48h MN-RET达峰值,且MN-RET呈现明显的剂量依赖性增加(r=0.984 9,P<0.01);重复染毒5d,FCM监测的MN-RET染毒2d后达到稳态水平。FCM检测的MN-RET峰值和稳态水平与常规显微镜观察的骨髓微核率有良好的相关性(r=0.9212,P<0.01)。结论:FCM小鼠外周血微核法稳定可靠,可以作为常规显微镜小鼠骨髓阅片法的替代方法,用于小鼠体内微核试验。OBJECTIVE：To overcome time-consuming,laborious,subjective and other shortcomings of the traditional microscopic examination method of micronuclei（MN）,we established flow cytometry（FCM） method for automatic analysis of micronuclei in mouse peripheral blood stained with anti-CD71-FITC,anti-CD61-PE and PI. METHOD：Mice received a single oral dose of 0,20,40,80 mg/kg cyclophosphamide and blood was sampled at different times before and after treatment.Additional mice were orally treated with 40 mg/kg cyclophosphamide for 5 consecutive days and blood was collected before and after treatment at 24 h interval.Blood cells were fixed in -80℃methanol and stained with anti-CD71-FITC,anti-CD61-PE,RNase and PI.After instrument was calibrated with malaria-infected erythrocytes as a biological standard,the frequencies of micronucleated CD71-positive reticulocytes were measured by FCM.RESULTS：The highest frequency of MN-RET was observed at 48 h after single treatment,and MN-RET showed dose-dependent accumulation（r=0.9849,P〈0.01）.These data acquired by FCM demonstrated that the steady-state of MN-RET was attained approximately 24 h after the second administration with 5 consecutive days treatment.Parallel analysis of micronucleus induction in peripheral blood by FCM and bone marrow using traditional microscopy-based method showed concordant results（r=0.9212,P〈0.01）.CONCLUSION：The three-color flow cytometric assessment of MN-RET in mouse peripheral blood can be considered as an acceptable substitute to microscopy-based analysis in mouse bone marrow micronucleus test in vivo.

关 键 词：CD71 流式细胞仪 网织红细胞 微核 环磷酰胺 

分 类 号：R99[医药卫生—毒理学]