摘要
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 clarify the relationship between the expression of pol β and DNA damage /repair inducedy by Benzo（a）pyrene （BaP）.Methods pol β wild-type cells （pol β ＋/＋）,pol β null cells （pol β-/-） and wild-type pol β overexpressed cells （pol β oe） which had the same genetic background were studied.Firstly,RT-PCR and Western blot targeting to pol β were carried out to measure the expression of pol β mRNA and protein in above three kinds of cells,then MTT test and single cell gel electrophoresis （comet assay） were used to compare cell viability and DNA damage/repair of the three kinds of cells when exposed to BaP.Results There was pol β deletion in pol β-/-cells and the level of pol β mRNA and protein in pol β oe cells was twice higher than that in pol β ＋/＋ cells.BaP could induce DNA damage and reduce cell viability,when compared with pol β ＋/＋ cells,IC_ 50 of pol β-/-cells was remarkably lower,DNA was prone to damage and more difficult to be repaired,on the other hand,IC 50 of pol β oe cells was obviously higher and the damage effect on DNA was weaker and prone to be repaired.Conclusion Pol β played an important role in the repair of DNA damage induced by BaP,deficiency of pol β could decrease the DNA repair capability of cells,and overexpression of pol β could help cells response to DNA damage and protect cells from death in a certain degree.
出处
《卫生研究》
CAS
CSCD
北大核心
2010年第1期13-18,共6页
Journal of Hygiene Research
基金
国家自然科学基金资助项目(No.30872079)
