›› 2011, Vol. 23 ›› Issue (2): 128-133.doi: 10.3969/j.issn.1004-616x.2011.02.011

• 技术与方法 • 上一篇    下一篇

基于三色流式细胞术的小鼠外周血微核试验方法的建立

周长慧1,王 征1,王庆利2,杨琛懋1,常 艳1﹡   

  1. 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
  • 收稿日期:2010-11-04 修回日期:2010-11-29 出版日期:2011-03-30 发布日期:2011-03-30
  • 通讯作者: 常 艳

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

ZHOU Chang-hui1,WANG Zheng1,WANG Qing-li2,YANG Chen-mao1,CHANG Yan1,*   

  1. 1. National Shanghai Center for New Drug Safety Evaluation & Research, Shanghai Institute of Pharmaceutical Industry, Shanghai 201203; 2. Center for Drug Evaluation, State Food and Drug Administration, Beijing 100038, China
  • Received:2010-11-04 Revised:2010-11-29 Online:2011-03-30 Published:2011-03-30
  • Contact: CHANG Yan

摘要: 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80 mg/kg环磷酰胺(CP),于不同时间点收集外周血;另一批小鼠给予CP 40 mg/(kg·d)经口灌胃,连续染毒5 d,分别于给药前和每次给药后24 h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48 h 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5 d,FCM监测的 MN-RET染毒2 d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=0.921 2, 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。

关键词: CD71, 流式细胞仪, 网织红细胞, 微核, 环磷酰胺

Abstract: 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.

Key words: CD71, flow cytometry, reticulocyte, micronucleus, cyclophosphamide