基于L5178Y细胞的Pig-a基因突变试验方法学研究

目的:采用不同作用机制化合物研究基于L5178Y细胞体外Pig-a基因突变试验方法。方法:使用不同浓度范围的马兜铃酸I(aristolochic acid I,AAI)、N-亚硝基二乙胺(N-nitrosodiethylamine,NDEA)、甲磺酸甲酯(methyl methanesulfonate,MMS)、环磷酰胺(cyclophosphamide,CP)、秋水仙素(colchicine,COL)、丝裂霉素C(mitomycin C,MMC)、糖精、乙烯利、苯并芘benzoa pyrene,B(a)P]和4-硝基喹啉-N-氧化物(4-nitroquinoline N-oxide,4-NQO)共10种化合物,分别与L5178Y细胞作用一段时间后表达8 d,细胞经APC抗CD45与PE抗CD90.2抗体孵育后使用流式细胞术收集100万个细胞并识别表型为CD90-/CD45⁺突变细胞,并计算突变率。结果:致突变剂AAI、NDEA、MMS、MMC、B(a)P、4-NQO分别在无或有代谢活化条件下导致L5178Y细胞Pig-a基因突变率显著性升高,而需代谢活化的致突变剂CP、整倍体诱变剂COL、非遗传毒性致癌物乙烯利和糖精的试验结果为阴性。结论:基于L5178Y细胞的体外Pig-a基因突变检测方法可在无及有代谢活化条件下有效检出致突变剂,特异性和可靠性较高,在药物基因突变风险评价及机制研究领域具有不可或缺的价值。

Pig-a gene mutation test methodology research based on L5178Y cells

Objective:Different mechanisms of action compounds were studied based on Pig-a gene mutation test in L5178 Y cells in vitro.Methods:A total of 10 compounds,including aristolochic acid I(AAI),N-nitrosodiethylamine(NDEA),methyl methanesulfonate(MMS),cyclophosphamide(CP),colchicine(COL),mitomycin C(MMC),saccharin,ethephon,benzopyreneB(a)P]and 4-nitroquinoline-N-oxide(4-NQO)respectively at different concentration ranges were treated with L5178 Y cells for a period and subsequently expressed 8 days.The cells were collected and incubated with APC-anti-CD45 and PE-anti-CD90.2,and cells with a phenotype as CD90-/CD45⁺were recognised by flow cytometry as mutant in a total of million cells for calculating the mutation rates.Results:Mutagens AAI,NDEA,MMS,MMC,B(a)P and 4-NQO induced significant increases on the Pig-a gene mutation rates in L5178 Y cells;while the results for CP as mutagen only after metabolism,euploid clastogen COL,non-genotoxic carcinogens ethephon and saccharin were negative.Conclusion:The in vitro Pig-a gene mutation assay based on L5178 Y cells can effectively detect mutagens in both absence and presence the metabolic activation,and has showed high specificity and reliability.It is indispensable in future risk evaluation and underlying mechanism research of the drug induced gene mutation.