高氧暴露和小分子干扰RNA对A549细胞核转录相关因子-2与NAD(P)H:醌氧化还原酶1表达的影响及其与细胞凋亡的关系  

Effects of hyperoxia exposure and small interfering RNA on the expressions of nuclear factor-erythroid 2-related factor 2 and NAD(P)H quinone oxidoreductase 1 enzyme in A549 cells and their relationship with apoptosis

作　　者：翁博雯 李帅 蔡成[1] 孙俊芳 张敏 成利花 Weng Bowen;Li Shuai;Cai Cheng;Sun Junfang;Zhang Min;Cheng Lihua(Department of Neonatology,Children's Hospital Affiliated to Shanghai Jiaotong University,Shanghai Children's Hospital,Shanghai 200062,China;Department of Neonatology,Fuyang Women and Children's Hospital(Fuyang Sixth People's Hospital),Fuyang 236000,Anhui Province,China)

机构地区：[1]上海交通大学附属儿童医院上海市儿童医院新生儿科,200062 [2]阜阳市妇女儿童医院(阜阳市第六人民医院)新生儿科,安徽阜阳236000

出　　处：《中华实用儿科临床杂志》2020年第21期1663-1667,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金面上项目(81571467)。

摘　　要：目的研究高氧暴露和小分子干扰RNA(siRNA)对A549细胞中细胞核转录相关因子2(Nrf2)、NAD(P)H:醌氧化还原酶1(NQO1)表达的影响,探讨其在高氧肺损伤中的作用及其与细胞凋亡的关系。方法将体外培养A549细胞随机分为4组:Ⅰ组为无干扰空气组;Ⅱ组为无干扰高氧组;Ⅲ组为Nrf2 siRNA转染空气组;Ⅳ组为Nrf2 siRNA转染高氧组;将Ⅱ组、Ⅳ组持续暴露于常压高浓度氧(950 mL/L O2,50 mL/L CO2)中;Ⅰ组、Ⅲ组仍置于50 mL/L CO2培养箱中。通过预实验从目的基因靶点siRNA-1、siRNA-2、siRNA-3(分别加入对应脂质体复合物)中筛选出抑制Nrf2效率最高的Nrf2 siRNA,应用实时荧光定量聚合酶链反应(qPCR)、Western blot分别检测4组Nrf2、NQO1的mRNA及蛋白表达水平,应用免疫荧光和激光共聚焦显微镜分析干扰Nrf2后A549细胞中Nrf2、kelch样ECH相关蛋白1(Keap1)、抗氧化物反应元件蛋白的分布,观察各组细胞凋亡情况。结果1.Nrf2 siRNA可抑制Nrf2表达,siRNA-1组、siRNA-2组、siRNA-3组Nrf2 mRNA表达均被抑制,其中siRNA-1组的抑制效率最高(80.57%)。2.Ⅱ组Nrf2、NQO1 mRNA的相对表达量分别为4.553±0.498和5.866±0.582,与Ⅰ组相比,mRNA及蛋白表达量显著增高,细胞凋亡率[(21.67±0.75)%]增加,差异均有统计学意义(均P<0.01)。3.Ⅳ组Nrf2、NQO1 mRNA的相对表达量分别为0.937±0.057和0.789±0.058,与Ⅱ组相比,mRNA及蛋白表达显著减弱,细胞凋亡率[(35.83±0.42)%]进一步增高,差异均有统计学意义(均P<0.01)。结论高氧暴露和siRNA导致A549细胞中Nrf2和NQO1表达异常,提示Nrf2和NQO1参与高氧肺损伤的发病过程;Nrf2、NQO1可能在高氧所致肺损伤及细胞凋亡中发挥保护作用。Objective To analyze the effect on the expressions of nuclear factor-erythroid 2-related factor 2(Nrf2)and NAD(P)H quinone oxidoreductase 1 enzyme(NQO1)in A549 cells exposed to hyperoxia and interfered by small interfering RNA,and to investigate the role of Nrf2 and NQO1 in hyperoxia-induced lung injury as well as their relationship with apoptosis.Methods A549 cells were gained by serial sub cultivation in vitro and then randomly divided into 4 groups:the air group without interference(group Ⅰ),the hyperoxia group without interference(group Ⅱ),the air group transfected with Nrf2 siRNA(group Ⅲ),and the hyperoxia group transfected with Nrf2 siRNA(group Ⅳ).The hyperoxia groups(Ⅱ,Ⅳ group)were continuously exposed to an atmosphere containing a high concentration of oxygen(950 mL/L O2,50 mL/L CO2),while the air groups(group Ⅰ,Ⅲ)were still placed in the incubator with 50 mL/L CO2.In the pre-experiment,cells were transduced with a mixture of siRNA-1,siRNA-2,and siRNA-3.Then the siRNA with the highest efficiency for repressing Nrf2 expression was used for subsequent experiments.The mRNA and protein expression levels of Nrf2 and NQO1 in the 4 groups were detected by quantitative real-time polymerase chain reaction(qPCR)and Western blot.The distribution of Nrf2,Kelch-like ECH-associated protein 1(Keap1)and antioxidant response element(ARE)proteins in A549 cells after interference with Nrf2 was analyzed by immunofluorescence and confocal laser scanning microscope,and the cell apoptosis of the 4 groups were observed.Results(1)Nrf2 siRNA significantly down-regulated the mRNA expression of Nrf2 in the groups siRNA-1,siRNA-2 and siRNA-3,and the inhibition efficiency of group siRNA-1 was the highest(80.57%).(2)The re-lative mRNA expression levels of Nrf2 and NQO1 in the group Ⅱ were 4.553±0.498 and 5.866±0.582,respectively.The mRNA and protein expression of Nrf2 and NQO1 and the cell apoptosis rate[(21.67±0.75)%]in the hype-roxia group were significantly higher than those in the group Ⅰ(all P<0.01).(3)The r

关 键 词：高氧肺损伤 核转录相关因子-2 NAD(P)H:醌氧化还原酶1 早产 

分 类 号：R722.6[医药卫生—儿科]