DNA修复相关基因SNP与辐射致离体血微核率的相关性研究

Research on the Correlation Between SNP Associated with DNA Repair Genes and Blood Micronucleus Frequency in Vitro Induced by Radiation

目的分析DNA修复相关基因单核苷酸多态性(single nucleotide polymorphism,SNP)与辐射致离体血微核率(micronucleus frequency,MNF)的关系,为寻找辐射易感性标志物提供理论依据。方法采集75例研究对象的外周血并分为乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)抗凝血样本和肝素抗凝血样本。提取EDTA抗凝血样本的DNA,设计8种基因11个SNP的多重聚合酶链式反应(polymerase chain reaction,PCR)引物和单碱基延伸引物,通过PCR扩增、延伸,应用DP-TOF型飞行时间质谱检测系统对SNP位点进行基因分型。将肝素抗凝血样本分为两份,一份行1.0Gy X线照射为辐照组,另一份未行照射为对照组,采用胞质分裂阻断微核实验对两组外周血淋巴细胞进行MNF测定。结果75份样本11个SNP位点均分型成功,检出率100.0%。辐照后,外周血淋巴细胞MNF升高(1.919‰±0.642‰vs.0.177‰±0.107‰,t=22.925,P<0.001)。X线交叉互补修复基因1(X-ray repair cross complementing 1,XRCC1)rs25487与辐照组MNF相关(P<0.01),且TC基因型个体MNF高于CC基因型(2.270‰±0.597‰vs.1.695‰±0.588‰,P<0.01);XRCC1rs1799782与辐照组MNF相关(P<0.05)且AA基因型个体MNF高于GG基因型(1.943‰±1.078‰vs.1.790‰±0.461‰,P<0.05)。X线交叉互补修复基因3(X-ray repair cross complementing 3,XRCC3)rs1799794与辐照组MNF相关(P<0.01)且TT和CC基因型个体MNF均高于TC基因型(2.123‰±0.707‰vs.1.680‰±0.411‰,P<0.01;1.979‰±0.763‰vs.1.680‰±0.411‰,P<0.05)。8-羟基鸟嘌呤糖苷酶(8-oxoguanine DNA glycosylase,hOGG1)基因rs1052133与辐照组MNF相关(P<0.05),且CC基因型个体MNF高于CG基因型(2.260‰±0.435‰vs.1.824‰±0.544‰,P<0.05),其余基因SNP位点与辐照组MNF的相关性无统计学意义(P>0.05)。结论辐射可导致MNF升高,rs25487,rs1799782,rs1799794和rs1052133的基因型与辐射致MNF有关联,可作为潜在的辐射敏感标志物。

Objective To analyze the relationship between single nucleotide polymorphism(SNP)associated with DNA repair genes and blood micronucleus frequency(MNF)in vitro induced by radiation,so as to provide a theoretical basis for radiation susceptibility biomarkers.Methods Peripheral blood samples were collected from75 subjects and divided into ethylene diamine tetraacetic acid(EDTA)anticoagulant samples and heparin anticoagulant samples.DNA was extracted from EDTA anticoagulant samples,multiple polymerase chain reaction(PCR)primers and single base extension primers were designed for 11 SNP of 8 genes,and PCR amplification and extension were performed,SNP locus were genotyped using the DP-TOF time-of-flight mass spectrometry detection system.Heparin anticoagulant samples were divided into two groups,one irradiated with 1.0Gy X-ray was considered as the irradiation group and the other not irradiated was considered as the negative control group,and MNF in peripheral blood lymphocytes of two groups was determined by the cytoplasmic division blocking micronucleus assay.Results A total of 75 samples were successfully genotyped for 11 SNP locus,with a detection rate of 100.0%.After irradiation,the MNF of peripheral blood lymphocytes increased(1.919‰±0.642‰vs.0.177‰±0.107‰,t=22.925,P<0.001).X-ray repair cross complementing 1(XRCC1)rs25487 was correlated with the MNF in the irradiation group(P<0.01),and the MNF of TC genotype individuals was higher than that of CC genotype individuals(2.270‰±0.597‰vs.1.695‰±0.588‰,P<0.01);XRCC1 rs1799782 was correlated with the MNF in the irradiation group(P<0.05),and the MNF of AA genotype individuals was higher than that of GG genotype individuals(1.943‰±1.078‰vs.1.790‰±0.461‰,P<0.05).X-ray repair cross complementing 3(XRCC3)rs1799794 was correlated with the MNF in the irradiation group(P<0.01),and both uthe MNF of TT and CC genotype individuals were higher than that of TC genotype individuals(2.123‰±0.707‰vs.1.680‰±0.411‰,P<0.01;1.979‰±0.763‰vs.1.680‰±0.411‰,P<0.05).8-oxoguanine DNA glycosylase(hOGG1)gene rs1052133 was correlated with the MNF in the irradiation group(P<0.05),and the MNF of individuals with CC genotype individuals was higher than that of individuals with CG genotype(2.260‰±0.435‰vs.1.824‰±0.544‰,P<0.05),the correlation between SNP locus of other genes and MNF in the irradiation group was not statistically significant(P>0.05).Conclusion Radiation could lead to an increase in MNF;rs25487,rs1799782,rs1799794 and rs1052133 are associated with the MNF induced by radiation,which may become potential radiosensitive biomarkers.