中文摘要：

目的：建立野生型Keap1过表达的H460细胞株降表达Nrf2,检测Nrf2-ARE信号通路对肿瘤细胞耐药性的影响。方法：H460细胞转染mKeap1-pEGFP后经过长期筛选得到稳定表达Keap1蛋白质的细胞株,通过Western blotting和荧光定量PCR的方法进行检测;并通过多次继代培养,细胞株H460-N5稳定表达mKeap1,Nrf2及其调控基因均显著降表达;用MTS法检测抗癌药物奥沙利铂、阿霉素和依托泊苷对细胞增殖抑制率。结果：H460细胞转染mKeap1-pEGFP后筛选建立Nrf2降表达的稳定细胞株H460-N5。MTS数据显示,与对照组相比,抗癌药物奥沙利铂、阿霉素和依托泊苷对H460-N5细胞的抗增殖作用更显著。当奥沙利铂和依托泊苷分别在93μmol/L和100μmol/L浓度作用于对照组细胞株H460-N0时,药物作用接近半数抑制率（IC50）;而在H460-N5细胞株中,两种抗癌药物的IC50分别为42μmol/L和30μmol/L。阿霉素对H460-N0细胞的IC50〉3 mg/L,而对H460-N5细胞的IC50约为2 mg/L。结论：Keap1过表达的H460-N5细胞Nrf2及调控基因显著降低并增敏抗癌药物奥沙利铂、阿霉素和依托泊苷对肿瘤细胞的增殖抑制作用。

英文摘要：

Objective： To maked a Nrf2 down-regulated cell line by over-expressing Keap1 in H460 cells to study the role of Nrf2 in drug resistance.Methods： Transfecting H460 cells with mKeap1-pEGFP and screennig for Keap1 expressing clones by Western blotting with antibodies against Nrf2,HO-1,NQO1 and AKR1C.The cell line with Keap1 over-expression was further confirmed by real-time PCR.The cytotoxicity of H460-N5 to anti-cancer drugs was evaluated by MTS assay.Results： MTS assay results showed the enhanced cytotoxicity of anticancer drugs（Oxaliplatin,Doxorubicin and Etopside） to the H460 cell line with keap1 overexpression compared to the control cell line.In H460-N0 cells,the IC50 values of Oxaliplation and Etopside were 93 μmol/L and 100 μmol/L respectively whereas the IC50 values of the two drugs were 42 μmol/L and 30 μmol/L correspondingly in H460-N5 cells.The IC50 value of Doxorubicin to H460-N0 cell was above 3 mg/L,but that to H460-N5 cell was about 2 mg/L.Conclusions： A Nrf2 down-regulated cell line H460-N5 and a control cell line with GFP over-expression have been identified.Down-regulation of Nrf2 enhanced the cytotoxicity of Oxaliplatin,Doxorubicin and Etopside.