癌变·畸变·突变

• 技术与方法 • 上一篇    下一篇

体内Pig-a基因突变试验高通量方法联合验证研究

 铭,周长慧,常 艳*,王 征,涂宏刚,李 婕   

  1. ( 中国医药工业研究总院国家上海新药安全评价研究中心,上海 201203 )
  • 收稿日期:2013-04-22 修回日期:2013-07-03 出版日期:2013-09-30 发布日期:2013-09-30
  • 通讯作者: 常 艳,E-mail:ychang@ ncdser.com
  • 作者简介:张 铭 (1986- ),男,山东人,硕士研究生,研究方向:遗传毒理学。E-mail:aiguozhe0301@163.com
  • 基金资助:

    国家“十二五”重大专项基金资助项目 (2012ZX09505-001-003),上海市科学技术委员会上海研发公共服务平台建设专项(11DZ2292100)

Interlaboratory transferability study of the Pig-a mutation assay with immunomagnetic enrichment

ZHANG Ming,ZHOU Chang-hui,CHANG Yan*,WANG Zheng,TU Hong-gang,LI Jie   

  1. (National Shanghai Center for New Drug Safety Evaluation & Research, China State Institute of Pharmaceutical Industry, Shanghai 201203, China)
  • Received:2013-04-22 Revised:2013-07-03 Online:2013-09-30 Published:2013-09-30
  • Contact: CHANG Yan,E-mail:ychang@ ncdser.com

摘要:

 目的: 建立大鼠磷脂酰肌醇聚糖A类 (Pig-a)基因突变试验高通量方法,验证该方法的重复性,并探索将此方法与流式体内微核整合至同一个试验中的可能性。方法:将20只雄性SD大鼠按照体质量随机分为4组:溶剂对照组 (PBS,pH=6.0)、N-乙基- N-亚硝基脲 (N-ethyl-N-nitrosourea,ENU)低剂量组 (10 mg/kg)、ENU中剂量组 (20 mg/kg)和ENU高剂量组 (40 mg/kg),给药容量均为10 mL/kg,经口灌胃给药,每天1次,连续给药3 d。分别于给药前1天、首次给药后第14和30天颈静脉取血,进行Pig-a基因突变分析;首次给药后第4天颈静脉取血,采用流式细胞仪进行微核检测。结果:在首次给药后第14和30天,3种剂量 (10、20和40 mg/kg)下的ENU均导致大鼠外周血RBCCD59-和RETCD59-频率显著增加 (P<0.05),Pig-a基因突变结果与之前的基础型试验得到的结果类似,分析速率和分析细胞的数目得到显著提升;3个剂量的ENU均导致明显的微核率升高,与对照组比较,差异均具有统计学意义 (P<0.05)。结论:本研究成功建立了大鼠体内Pig-a基因突变高通量试验方法,该试验具有较好的重复性,并提示该试验可以和微核试验结合到同一个试验中。

关键词:  N-乙基-N-亚硝基脲, Pig-a基因突变, 免疫磁珠筛选, 微核, 流式细胞术

Abstract:

OBJECTIVE: To evaluate the reproducibility and transferability of the erythrocyte(RBC) based Pig-a mutation assay and explore the potential of combining this assay and micronucleus(MN) analyses into one study. METHODS20 rats were randomly assigned to be treated with PBS and different doses of (10,20 and 40 mg/kg) N-ethyl-N-nitrosourea (ENU) for 3 consecutive days by oral gavage. Jugular blood samples were collected on days -1 (the day before administration),14,and 30 and evaluated for Pig-a mutantion frequencies in whole peripheral blood RBCs and reticulocytes (RETs). Day 4 samples were scored for micronucleated reticulocyte frequencies. RESULTS While ENU-induced Pig-a mutation frequencies were consistent with previously reported results,analysis rate and number of cells evaluated were dramatically increased. ENU also induced significant increases in MN-RET frequencies. CONCLUSION: The in vivo Pig-a mutation high-throughput assay was established successfully in rat. Results with ENU indicated that the new Pig-a scoring methodology was reproducible Pig-a mutation and MN analyses could be readily combined into one study.

 

Key words:  N-ethyl-N-nitrosourea, Pig-a mutation, immunomagnetic separation, micronucleus, flow cytometry