背景:近年来研究发现食管癌细胞中存在DNA 聚合酶β的突变.目的:观察互隔交链孢酚对NIH/3T3 细胞中DNA 聚合酶β基因序列的影响,研究互隔交链孢酚致细胞变异的机制.方法:用不同浓度(2,4,6,8 μmol/L)的互隔交链孢酚作用于NIH/3T3 细胞,并设置对照组.用RT-PCR 法从细胞总RNA中扩增出DNA 聚合酶β基因cDNA 序列,应用T-A 克隆技术,克隆至pGEM-T 载体后进行测序,分析其序列变化.结果与结论:2 μmol/L 互隔交链孢酚处理后的细胞内DNA 聚合酶β基因序列无任何变化.而4 μmol/L 组中DNA 聚合酶β基因有1 个位点发生突变,8 μmol/L 组和16 μmol/L 组中各有2 个位点发生突变,此3 组的突变存在相同的位点.结果证实,互隔交链孢酚可导致NIH/3T3 细胞中的DNA 聚合酶β发生点突变,互隔交链孢酚浓度越高,点突变的数量越多,且存在突变活跃位点.%BACKGROUND: The mutation of DNA polymerase β (DNA pol β) has been found in esophageal carcinoma cells. OBJECTIVE: To analyze the effect of alternariol on the sequence of DNA pol β in NIH/3T3 cell line and to study the mutation mechanism of alternariol METHODS: The NIH/3T3 cell line was injected with different concentrations of alternariol (2, 4, 6, 8 μmol/L) and the control group was set. DNA pol β gene was amplified from the total RNA of the cells using reverse transcription-PCR, then cloned with pGEM-T vector and sequenced with T-A cloning technique, and the sequence was analyzed. RESULTS AND CONCLUSION: The sequence of DNA pol β was not changed in 2 μmol/L alternariol group, and there was one mutation point in 4 μmol/L alternariol group and two mutation points in 8 and 16 μmol/L alternariol group. There was a same mutation point in these three groups. Alternariol can induce point mutation of DNA pol β gene in NIH/3T3 cell line. Higher concentration of alternariol, more points of DNA pol β gene mutation. The active mutation point exists.
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