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目的揭示聚合酶β(polβ)的表达水平与苯并[a]芘(BaP)诱导的DNA损伤效应的关系。方法采用3种具有相同遗传背景的小鼠胚胎成纤维细胞polβ野生型(polβ+/+)、polβ缺陷型(polβ-/-)和野生型polβ高表达型(polβoe)作为模型细胞。首先用RT-PCR和Western blot鉴定3种细胞中polβ在mRNA和蛋白质水平的表达情况,然后通过MTT试验和单细胞凝胶电泳(彗星试验)观察BaP作用下3种细胞的存活率及DNA损伤和修复效应。结果3种细胞在polβmRNA和蛋白表达水平上存在明显差异,polβ-/-细胞中polβ基因缺失,而polβoe细胞中polβ基因则较野生型polβ+/+细胞高出2倍以上;BaP能诱导3种细胞DNA的损伤以及细胞存活率的降低;与polβ+/+细胞相比,polβ-/-细胞的IC50更低,DNA损伤更严重且更加不易修复;反之,polβoe细胞的IC50更高,DNA损伤效应更弱,DNA修复速率加快。结论polβ在修复外源性致癌物BaP导致的DNA损伤中发挥着重要的作用,polβ基因缺陷使细胞DNA修复能力降低,而polβ基因的高表达则能帮助细胞应对DNA损伤,在一定程度上对细胞的存活起到了保护作用。
Objective To reveal the relationship between the expression level of polβ and the DNA damage induced by benzo [a] pyrene (BaP). Methods Three mouse embryonic fibroblasts with polβ wild type (polβ + / +), polβ - deficient (polβ - / -) and wild-type polβ oocytes with the same genetic background were used as model cells. First, RT-PCR and Western blot were used to detect the expression of polβ mRNA and protein in three kinds of cells. Then the cell viability and DNA damage of BaP were observed by MTT assay and single cell gel electrophoresis (comet assay) And repair effect. Results Polβ mRNA and protein expression levels were significantly different in three kinds of cells, polβ gene was absent in polβ - / - cells and polβ gene in polβoe cells was more than 2 times higher than that in wild type polβ + / + cells. BaP could induce 3 Polβ - / - cells have lower IC50, DNA damage is more serious and more difficult to repair; polβoe cells in contrast, IC50 higher, DNA Lesser lesion effect, DNA repair faster. Conclusion Polβ plays an important role in repair of DNA damage induced by exogenous carcinogen BaP. Polβ gene deficiency can reduce DNA repair ability, while high expression of polβ gene can help cells to deal with DNA damage. To a certain extent, Cell survival has played a protective role.