NRF2介导的还原应激在砷致HaCaT细胞恶性转化中的作用  

NRF2 mediated redox stress in arsenic induced human keratinocytes malignant transformation

作　　者：张婷[1] 姚广泽 陈慧婷 杨乾磊 安艳[1] Zhang Ting;Yao Guangze;Chen Huiting;Yang Qianlei;An Yan(School of Public Health,Medical College of Soochow University,Suzhou 215123,China;The First Clinical School of Medicine,Soochow University,Suzhou 215123,China)

机构地区：[1]苏州大学苏州医学院公共卫生学院,苏州215123 [2]苏州大学第一临床医学院,苏州215123

出　　处：《卫生研究》2024年第5期763-770,789,共9页Journal of Hygiene Research

基　　金：国家自然科学基金(No.81872646,81811540034,81573173,82381240027);国家级大学生创新创业训练计划项目(No.202210285068Z);2022年秦惠与李政道中国大学生见习进修基金。

摘　　要：目的 了解核转录因子E2相关因子2(nuclear transcription factor E2-related factor 2,NRF2)介导的还原应激在砷致人皮肤角质形成细胞(HaCaT细胞)恶性转化中的作用。方法 将HaCaT细胞及荧光标记线粒体谷胱甘肽的HaCaT(Mito-Grx1-roGFP2 HaCaT)细胞分别在含0.0和1.0μmol/L NaAsO_(2)的培养基中培养到35代,建立细胞恶性转化模型。在第0代、早期(第1、7和14代)和晚期(第21、28和35代)分别检测HaCaT细胞和线粒体中还原型谷胱甘肽/氧化型谷胱甘肽(glutathione/oxidized glutathione,GSH/GSSG)和还原型辅酶II/氧化型辅酶II(coenzyme II/oxidized coenzyme II,NADPH/NADP^(+))比值;Mito-Grx1-roGFP2 HaCaT细胞倍增时间、细胞迁移能力、软琼脂克隆形成能力及线粒体GSH/GSSG比值。恶性转化细胞转染NRF2小干扰RNA(siRNA)沉默NRF2表达后,检测NRF2表达水平及上述各指标。结果 与对照组比较,1.0μmol/L NaAsO_(2)染毒HaCaT细胞GSH/GSSG比值在第1代和第7代明显降低,第21代以后显著升高;第1、14、21、28和35代NADPH/NADP^(+)比值显著升高。线粒体GSH/GSSG水平在第1~35代显著增加,第1、7、21、28和35代NADPH/NADP^(+)水平显著升高。1.0μmol/L NaAsO_(2)染毒Mito-Grx1-roGFP2 HaCaT细胞35代细胞倍增时间明显缩短;48 h后的划痕距离明显缩小,细胞迁移率明显增加;且能够形成明显的克隆集落。Mito-Grx1-roGFP2 HaCaT细胞线粒体内的GSH/GSSG比值在第1代明显降低,从第7代后显著增加(P<0.05)。用siRNA沉默NRF2表达后,与转染对照组比较,转染组过氧化氢和超氧化物水平均增加;细胞内和线粒体中的GSH/GSSG比值、NADPH/NADP^(+)比值,Mito-Grx1-roGFP2 HaCaT细胞线粒体内的GSH/GSSG比值均降低;细胞倍增时间增加,细胞迁移能力和软琼脂克隆形成能力显著降低(P均<0.05),恶性转化被逆转。结论 砷染毒HaCaT细胞早期(第1、7和14代)为氧化应激,NRF2持续高表达;晚期(第21、28和35代)NRF2诱导还原应激,导致恶性转化。OBJECTIVE To explore the role of nuclear transcription factor E2-related factor 2(NRF2)-mediated reductive stress in arsenite induced malignant transformation in human keratinocytes.METHODS HaCaT cells and fluorescent labeled mitochondrial glutathione HaCaT cells(Mito-Grx1-roGFP2 HaCaT) were cultured to 35 passages in medium containing 0.0 and 1.0 μmol/L NaAsO_(2) to establish a model of malignant transformation of cells.Cellular and mitochondrial reduced glutathione/oxidized glutathione(GSH/GSSG) and reduced coenzyme II/oxidized coenzyme II(NADPH/NADP~+) ratios were measured in HaCaT cells.Cell doubling time,cell migration ability,soft agar clone formation ability and GSH/GSSG at different times in the 0 passage,the early stage(1st,7th and 14th passages) and later stage(21st,28th and 35th passages) were measured in Mito-Grx1-roGFP2 HaCaT cells.NaAsO_(2) induced malignant transformation cells were transfected with NRF2 siRNA,and detected the expression level of NRF2 and the redox-related indexes and malignant transformation indexes.RESULTSCompared with the control group,the GSH/GSSG ratio in 1.0 μmol/L NaAsO_(2) treated HaCaT cells significantly decreased in the 1st and 7th generations,but significantly increased after the 21st generation,and the NADPH/NADP~+ ratio significantly increased in the 1st,14th,21st,28th and 35th generations;The levels of GSH/GSSG in mitochondria significantly increased from 1st to 35th generation,and the levels of NADPH/NADP~+ in mitochondria significantly increased at 1st,7th,21st,28th and 35th generation.After continuous treatment of Mito-Grx1-roGFP2 HaCaT cells with 0.0 or 1.0 μmol/L NaAsO_(2) to 35 passages,the doubling time of cells treated with 1.0 μmol/L NaAsO_(2) was significantly shortened,the cell migration rate was increased greatly,and more clones with larger volumes than the control cells formed.The GSH/GSSG ratio in mitochondria of Mito-Grx1-roGFP2 HaCaT cells showed a significant decrease in the 1st generation and increased from the 7th generation onwards(all P<0.05)

关 键 词：亚砷酸钠 核转录因子E2相关因子2 还原应激 恶性转化 HACAT细胞 

分 类 号：R114[医药卫生—卫生毒理学]