hOGG1基因高表达A549细胞株的构建及其对氧化应激的影响

The construction of A549 cell line with over expression of hOGG1 and effect on DNA damage and repair

目的 构建人8-羟基鸟嘌呤-DNA糖苷酶（hOGG1）基因高表达细胞株,并初步探讨该细胞株对DNA氧化损伤与修复作用的影响.方法 提取人血液淋巴细胞总RNA,设计hOGG1特异性引物,RT-PCR扩增后构建pcDNA3.1（＋）/Myc-His A/hOGG1载体.经EcoRI和KpnI酶切,凝胶电泳及DNA测序证实建立成功后,稳定转染A549细胞,Western blot法检测转染阳性细胞hOGG1蛋白表达的改变.以构建的hOGG1基因高表达细胞株为研究对象,H2O2为受试物,彗星试验检测比较各组细胞对抗损伤和修复能力的情况.结果 成功构建pcDNA3.1（＋）/Myc-His A/hOGG1质粒并转染目的 细胞,Western blot法检测转染靶细胞中hOGG1蛋白表达比空白组和阴性对照组明显增加（P〈0.05）.相同H2O2浓度下,转染细胞彗星细胞率和Olive Tail Moment明显低于空白组和阴性对照组,且修复完成率高,修复时间缩短（P〈0.05）,提示细胞对抗DNA损伤和修复能力均显著增高P〈0.05）.结论 hOGG1基因的高表达可确切减轻细胞氧化损伤敏感性并提升DNA的修复能力.hOGG1基因高表达细胞株的成功构建,为基因水平的靶向治疗研究提供了一种有效手段.

Objective This study was conducted to establish A549 cell line with over expression of human 8 - oxaguanine - DNA glycosylase （ hOGG1 ）, and to explore the effect on DNA damage and repair, Methods The whole hOGG1 coding sequence was amplified by RT - PCR from total RNA and cloned into eukaryotic expression vector pcDNA3. 1 （ ＋ ）/Mye - His A. Stable transfection A549 cell line was constructed by transfeeting the recombinants into cells. Successful establishment was detected by Western blot for hOGG1 protein. Blank control cells, negative control and transfeeted cells were exposed to different concentration of hydrogen peroxide, Part of cells were tested respectively about Comet cell rate and Olive Tail Moment to evaluate the damage degree of cells DNA, and part of cells were incubated for 0, 60, 120 and 180 min after the oxidants removed. The same indexes were tested by modified comet assay, Results Results showed the sequence of hOGG1 matched that provided in gene bank and construction of eukaryotic expression vector pcDNA3.1 （ ＋ ）/Mye - His A - hOGG1 was successful. The expression of hOGG1 in stable transfeeted A549 - T cells was significantly higher than that in blank control cells and negative control cells （ P 〈 0.05 ）. Transfeeted cells showed stronger resistance to DNA oxidative damage and significantly increased the ability of DNA repair （P 〈 0.05 ）. No statistical significance on indications between blank control cells and negative control （P 〉 0. 05 ）. Conclusion The construction of eukaryotic expression vector pcDNA3.1 （ ＋ ）/ Myc - His A/hOGG1 provides a method for biological targeted therapy. The up - regulated expression of hOGG1 gene could increase the resistance to DNA damage and increase the ability in DNA repair in A549 cells exposed to oxidants.