基于L5178Y细胞体外Pig-a基因突变试验方法的建立与初步探索

利用小鼠淋巴瘤细胞L5178Y tk+/--3.7.2C和阴性化合物Glc、NaCl及阳性化合物EMS、ENU、4-NQO、B(a)P建立并验证基于哺乳动物细胞的体外Pig-a基因突变检测方法。计算细胞相对倍增速率评价细胞毒性,抗体标记突变型细胞确定流式检测模板,L5178Y细胞经EMS处理后第4、8、12、16、20天检测Pig-a基因突变频率,确定最大突变频率发生时间点,免疫荧光技术检测CD90蛋白在细胞中的定位情况,采用PCR方法进行突变位点分析。结果表明(:1)Glc、NaCl、EMS、ENU、B(a)P、4-NQO所设浓度组RPD均大于50%。(2)Pig-a基因突变频率在给药后第8天出现峰值。Glc和NaCl致Pig-a基因突变频率均小于200×10-6,各浓度组与溶剂对照组间不存在显著性差异(P>0.05),EMS、ENU、B(a)P、4-NQO均可引起Pig-a基因突变频率增加,且与溶剂对照组相比存在显著性差异。(3)免疫荧光成像显示突变型细胞表面无CD90蛋白,野生型细胞正常表达CD45,CD90蛋白。(4)基因突变位点检测显示存在G→C、A→C、C→T三种突变类型。基于小鼠淋巴瘤L5178Y细胞分别在有无S9代谢活化条件下成功建立体外Pig-a基因突变的遗传毒性检测方法,旨为化合物体外遗传毒性评价或药物研发早期遗传毒性筛选提供新方法。

Establishment and Preliminary Exploration of in vitro Pig-a Gene Mutation Assay Based on L5178Y Cells

The objective of this work isto establish and validatea method of detecting Pig-a gene mutation in mammalian cells based on mouse lymphoma cells L5178Y tk+/--3.7.2C with negative compounds Glc,NaCl and positive compounds EMS,ENU,4-NQO,and B(a)P.Cytotoxicity evaluation was performed by calculating the relative doubling rate of the cells,and the antibody-labeled mutant cells were used to determine the flow detection template.The frequency of Pig-a gene mutation was detected on the 4th,8th,12th,16th and 20th day after EMS treatment,and the time point of the maximum mutation frequency was determined.The location of CD90 protein in the cells was detected by immunofluorescence technique,and the PCR was used for mutation site analysis.Results are as following:1)The RPD of the concentration groups set by Glc,NaCl,EMS,ENU,B(a)P,and 4-NQO were all>50%.2)The frequency of mutation of the Pig-a gene peaked on the 8th day after administration.The mutation frequency of Glc and NaCl in Pig-a gene was<200×10-6.There was no significant difference between each concentration group and solvent control group(P>0.05).EMS,ENU,B(a)P,and 4-NQO resulted intheincrease in the mutationfrequency of the Pig-a gene,and there was a significant difference compared with the solvent control group.3)Immunofluorescence imaging showed that there was no CD90 protein on the surface of mutant cells,and wild type cells normally expressed CD45 and CD90 proteins.4)Detection of gene mutation sites revealed the presence of 3mutation types:G→C,A→C,and C→T.In conclusion,this study successfully establishes a method of detecting the genotoxicity of in vitro Pig-a gene mutation in mouse lymphoma L5178Y cells with/without of S9 metabolic activation,providing new approach in vitro genotoxicity evaluation or screening genotoxicity in the early development stage of drugs.