流式细胞术检测体外微核方法的验证  

Validation of flow cytometry in vitro micronuclear assay

作　　者：欧红梅 周长慧 涂宏刚 徐灵芝 常艳 

机构地区：[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

出　　处：《中国新药杂志》2015年第14期1590-1598,共9页Chinese Journal of New Drugs

基　　金：国家"重大新药创制"科技重大专项(2012ZX09505001-003);上海市研发公共服务平台专项(15DZ2290300)

摘　　要：目的:采用不同作用强度、不同作用机制的遗传毒性化合物及非遗传毒性化合物验证已建立的96孔板流式细胞术体外微核自动化检测方法。方法:试验分为+S9短时处理组(4 h)和-S9持续处理组(24 h),加样后24 h(5-氟尿嘧啶为48 h)收获细胞。验证的化合物分别选择3个不同浓度处理CHO-K1细胞,设置环磷酰胺(+S9组)或丝裂霉素C(-S9组)的阳性对照和溶剂对照。采用EMA和SYTOX green双色标记,流式细胞仪分析96孔板的微核率,并与常规平皿培养、人工阅片的细胞分裂阻滞法的双核微核结果进行比较。结果:人工阅片和流式细胞术检测验证化合物诱导CHO-K1细胞微核的结果一致。在有或无S9处理条件下,甲基磺酸甲酯、秋水仙素、依托泊苷和4-硝基喹啉-1-氧化物体外微核试验结果均为阳性;5-氟尿嘧啶和己烯雌酚仅在无代谢活化系统条件下,试验结果为阳性;苯并芘仅在代谢活化系统的情况下,试验结果为阳性;氯化钠、蔗糖和2-氨基蒽体外微核试验结果为阴性。结论:本试验进一步证实流式体外微核检测方法灵敏度高、特异性好,可代替人工阅片的双核微核方法,用于化合物遗传毒性早期筛选和遗传毒性评价。Objective： To validate the flow cytometric 96-well microplate-based in vitro micronuclear assay in CHO-K1 cells exposed to genotoxic compounds with different mechanisms or intensities,and to non genotoxic compounds. Methods： The tests contained treatments with and without metabolic activation. Each treatment included three different concentration groups,one positive control group and one negative control group. For the treatment with metabolic activation,CHO-K1 cells were treated in the S9 mix medium for 4 h. For the treatment without metabolic activation,cells were incubated continuously for 24 h. In all cases,after a total of 24 h [exception for the 5-fluorouracil（ 5-FU） recovery to 48 h]since initiation of the treatment,cells were processed for microscopic scoring or flow cytometric MN analysis. A flow cytometric method for scoring MN used EMA and SYTOX green to label the cells in 96-well microplates,and then compared with cytokinesis-block micronuclear assay in cell culture disks by microscopy. Results： The MN formation induced by both genotoxic agents and non genotoxic agents scored by microscopy and flow cytometry was consistent. Methyl methanesulfonate（ MMS）,colchicine（ COL）,etoposide（ ETO） and 4-Nitroquinoline-1-oxide（ 4NQO） resulted in significant increase of MN formation both in the absence of and presence of metabolic activation. 5-FU and diethylstilbestrol（ DIE） resulted in significant increase of MN formation only in the absence of metabolic activation. While benzo [α]pyrene（ B[α]P） re-sulted in significant increase of MN formation only in the presence of metabolic activation. Moreover,sodium chloride,sucrose and 2-aminoanthracene resulted in no significant increase of MN formation neither in the absence of nor in the presence of metabolic activation. Conclusion： The flow cytometry-based scoring method is sensitive and specific,so it would be alternative to the microscopy-based scoring method for the screening and detecting of genotoxic agents.

关 键 词：CHO-K1细胞 体外微核试验 流式细胞仪 人工阅片 方法验证 

分 类 号：R99[医药卫生—毒理学] R446[医药卫生—药学]