DNA聚合酶β表达水平与^(60)Coγ射线放射敏感性的研究

Exploration of Relationship between the Expression Level of DNA Polymerase β and ^(60)Coγ-ray Radiosensitivity

目的研究DNA聚合酶β(polβ)表达水平与60Coγ射线放射敏感性的关系,为增加临床放疗效果提供理论依据。方法以polβ野生型(polβ+/+)、polβ缺陷型(polβ-/-)及polβ高表达型(polβoe)小鼠胚胎成纤维细胞为研究对象,采用四氮唑盐(MTT)比色法和细胞集落形成实验探讨3种细胞对60Coγ射线的敏感性;同时通过2’,7’-二氯双氢荧光素双乙酸酯(DCFH-DA)荧光探针测定不同剂量60 Coγ射线照射后细胞内活性氧(ROS)含量。结果 MTT实验结果显示6,0Coγ射线照射后继续培养72h,3种细胞的存活率均随照射剂量的增加而下降,具有良好的剂量-效应关系(r+/+=-0.976、r-/-=-0.977、roe=-0.982,P均<0.05);相同剂量中,细胞存活率polβ-/-<polβ+/+<polβoe(P<0.05)。同样,3种细胞的集落数和集落形成率也均随照射剂量的增加而减少(P<0.05),相同剂量中,集落形成率polβ-/-<polβ+/+<polβoe(P<0.05)。此外,细胞内ROS水平均随60Coγ射线剂量的增加而增加(P<0.05);且相同剂量下,ROS水平polβ-/->polβ+/+>polβoe(P<0.05)。结论 60 Coγ射线引起的细胞死亡可能与ROS的产生及其导致的DNA氧化损伤有关,polβ高表达具有更强的DNA氧化损伤的修复作用,从而减少细胞死亡,增加细胞的辐照抗性,而polβ缺陷则可能增加细胞对60Coγ射线的敏感性。

Objective To explore the relationship between the expression level of DNA polymerase β（polβ） and 60Coγ-ray radiosensitivity and provide a basis on improving the efficiency of radiotherapy theoretically.Methods polβ wild-type cells（polβ＋/＋）,polβ null cells（pol β-/-） and polβ overexpressed cells（polβ oe） were applied as a model system.The radiosensitivity of 60Coγ-ray on the cell was detected by MTT assay and clone formation assay.The DCFH-DA fluorescent probe was used to examine the cellular ROS after 60Coγ-rays radiation.Results MTT assay showed that after radiation by 60Coγ-rays followed with 72 h incubation,the cell viabilities in the three kinds of cells decreased significantly with a dose-response relationship（r＋/＋=-0.976,r-/-=-0.977,roe=-0.982,P0.05）.In addition,the viability of polβ-/-cell was lower than those of other two kinds of cells at the same dose（P0.05）.Likewise,the colony number and colony formation rate in all tested cells also decreased after exposure to 60Coγ-rays.The ROS level in the three kinds of cells was enhanced after treatment with 60Coγ-ray,and the ROS level in polβ-/-cells was much higher than that in the other two kinds of cells（P0.05）.Conclusion Cell death caused by 60Coγ-ray may associated with the DNA oxidative damage mediated by ROS;Overexpression of polβ could protect against oxidative DNA damage,thus the cell apoptosis/death,thereby leading to reducing the radiosensitivity of 60Coγ-rays,while null of DNA polβ could increase radiosensitivity of 60Coγ-rays by compromising the DNA repair.