DNA氧化损伤修复基因hOGG_1高表达细胞株的建立

Establishment of a lung cancer cell line A549 highly expressing DNA oxidative damage related repair gene hOGG_1

目的联合pcDNA3.1(+)/Myc-HisA-hOGG1和pGL3promoter荧光质粒稳定转染人肺腺癌A549细胞获得hOGG1基因高表达细胞株,并初步探讨转染细胞生物学特性的变化。方法成功构建pcDNA3.1(+)/Myc-His A载体后,大量抽提阳性重组子并在Fu GENE6的介导下联合pGL3promoter荧光质粒转染A549细胞(转染组),同时设空白对照组(A549)和阴性对照组[PGL3+pcDNA3.1(+)/Myc-HisA转染的A549细胞]。生物荧光检测转染细胞hOGG1mRNA表达的改变,蛋白质免疫印迹法检测转染细胞hOGG1蛋白表达水平。将3组细胞于不同高氧条件下培养,相差显微镜观察形态学变化,彗星试验比较各组细胞对抗损伤和修复能力的情况,同时测定DNA氧化损伤标志物8-羟基脱氧鸟苷(8-OHdG)的变化。结果转染细胞荧光素酶稳定表达,蛋白印迹检测转染组hOGG1蛋白明显高于两对照组细胞,提示hOGG1基因高表达细胞株建立成功。相同高氧条件下,转染组形态学变化明显减轻,彗星细胞率和olive tail moment明显低于空白组和阴性对照组,修复完成率高,修复时间缩短(P<0.05),测定氧化损伤标志物8-OHdG明显下降,提示细胞对抗DNA损伤和修复能力均增高(P<0.05)。结论 hOGG1基因的高表达可减轻细胞对DNA氧化损伤的敏感性,提升细胞DNA的修复能力。

Objective To establish a human lung cancer cell line A549 highly expressing human 8-oxoguanine-DNA glycosylase（hOGG1）by co-transfecting pcDNA 3.1（＋）/Myc-HisA-hOGG1 and PGL3 promoter,and to observe the biological behavior of the transfected cells.Methods PcDNA3.1（＋）/Myc-His A-hOGG1 and PGL3 promoter were steadily co-transfected into A549 cells via mediation of Fu GENE 6（transfected group）;untransfected cells served as blank control and cells transfected with PGL3＋pcDNA3.1（＋）/Myc-HisA served as negative control.The hOGG1 mRNA expression in A549 cells was detected by Bio-luciferase activity and the hOGG1 protein expression by Western blotting analysis.Cells in the three groups were exposed to hyperoxia condition,and the morphological changes were observed by phase-contrast microscope.Comet cell rate and olive tail moment of cells were tested after different exposure periods.After exposure,the cells were incubated for 0,60,120,and 180 min,and the same indices were determined by modified comet assay;changes of DNA oxidative damage markers 8-hydroxy-desoxoguanosine（8-OHdG）was tested at the same time.Results The bio-luciferase activity was stable in the co-transfected cells.Western blotting analysis showed that the expression of hOGG1 protein in co-transfected A549-T cells was significantly higher than those in the other two groups,indicating the successful establishment of hOGG1 highly expressing cells.Under the same hyperoxia condition,the morphological changes of transfected cells were greatly alleviated,and the Comet cell rate and olive tail moment of cells were obviously lower than those of the other two groups（P0.05）.Transfected A549-T cells had significantly increased ability of DNA repair and shorter repair time compared with cells in the other two groups（P0.05）.Furthermore,the level of 8-OHdG in transfected A549-T cells was significantly lower than those of the other two groups under the same hyperoxia condition,indicating a significantly higher ability of DNA damage resisting and repair（P0.05）.Conclusion High hOGG1 expression can decrease the cell sensitivity to DNA damage and improve the repair ability of cells.