纳米二氧化硅致Nrf-2基因缺陷人永生化表皮细胞氧化损伤的研究  

Oxidative damage of Nrf-2 gene silenced human immortalized epidermal cells induced by silica nanoparticles

作　　者：陶功华[1] 肖萍[1] 孙静秋[1] 娄丹[1] 霍倩[1] 王彦琴[1] 李晨[1] 仲伟鉴[1] 帅怡[1] TAO Gong-hua XIAO Ping SUN Jing-qiu LOU Dan HUO Qian WANG Yan-qin LIChen ZHONG Wei- jian SHUAI Iq(Division of Chemical Toxicity Testing and Assessment, Shanghai Municipal Center for Disease Control and Prevention, Shanghai 200336, China)

机构地区：[1]上海市疾病预防控制中心化学品毒性检定所,上海200336

出　　处：《环境与职业医学》2017年第6期483-489,共7页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(编号:81302468);上海市卫生计生委资助项目(编号:20124080);上海市第四轮公共卫生三年行动计划"高端海外研修团队培养计划"项目(编号:GWTD2015S03)

摘　　要：[目的]探讨纳米二氧化硅(nano-SiO_2)致核因子E2相关因子2(Nrf-2)基因缺陷型人永生化表皮细胞的氧化应激损伤效应。[方法]采用RNA干扰技术构建人永生化表皮细胞(HaCaT)Nrf-2基因缺陷细胞模型,以终质量浓度(后称浓度)为2.5、5.0、10.0 mg/L的nano-SiO(215 nm粒径)分别染毒HaCaT细胞和Nrf-2基因缺陷细胞24 h,检测细胞内活性氧(ROS)、8-羟化脱氧鸟苷(8-oHdG)、丙二醛(MDA)、总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)等氧化应激指标,并对细胞总蛋白和核蛋白中Nrf-2蛋白的表达水平进行分析。[结果]5.0、10.0 mg/L nano-SiO_2可引起HaCaT细胞的活力明显降低(P<0.05),而2.5 mg/L nano-SiO_2开始即可引起Nrf-2基因缺陷细胞活力呈剂量依赖性降低(r=-0.914,P<0.05),Nrf-2基因缺陷细胞的活力均明显低于同浓度组的HaCaT细胞(P<0.05)。各浓度组细胞内ROS、8-oHdG和MDA水平均呈剂量依赖性增高,而T-SOD和GSH-Px的含量则呈剂量依赖性降低;与同浓度组HaCaT细胞相比,Nrf-2基因缺陷细胞中相关指标的变异程度更大,差异有统计学意义(P<0.05)。正常HaCaT细胞中,低剂量组总Nrf-2蛋白和低、中剂量组核Nrf-2蛋白的表达水平增高(P<0.05);而随着nano-SiO_2暴露浓度的进一步增高,两种蛋白的表达水平又呈降低的趋势。与HaCaT细胞的阴性对照比较,Nrf-2基因缺陷细胞中总Nrf-2蛋白表达水平下降,核Nrf-2蛋白表达水平未见差异。[结论]5.0 mg/L nano-SiO_2可体外诱导HaCaT细胞发生氧化应激性损伤,Nrf-2基因缺陷可通过改变细胞的抗氧化防御能力,增加HaCaT细胞对nano-SiO_2的敏感性。[ Objective ] To investigate the oxidative damage of nuclear factor E2-related factor 2 （Nrf-2） gene silenced human immortalized epidermal cells induced by silica nanoparticles （nano-SiO2）. [ Methods ] RNA interference technology was used to construct a Nrf-2 gene silenced cell model of human immortalized epidermal cells （HaCaT） （HaCaT-SiRNA-Nrf2）. Then the HaCaT and HaCaT-SiRNA-Nrf2 cells were exposed to different final concentrations （2.5, 5.0, and 10.0 mg/L） of nano-SiO2 （15 nm） for 24 h. Cellular reactive oxygen species （ROS）, 8-hydroxy-2- deoxyguanosine （8-oHdG）, malondialdehyde （MDA）, total superoxide dismutase （T-SOD）, and glutathione peroxidase （GSH-Px） were detected for each group. Western blot was used to detect total protein and nuclear protein expression levels of Nrf-2. [ Results ] The cell viability of HaCaT ceils decreased significantly in the 5.0 and 10.0mg/L nano-SiO2 groups （P〈0.05）, but 2.5, 5.0, and 10.0 mg/L nano-SiO2 decreased the viability of HaCaT-SiRNA-Nrf2 cells in a dose-dependent manner （r=-0.914, P 〈 0.05）,and the viabilities of HaCaT-SiRNA-Nrf2 cells were significantly lower in all dose groups compare with those of HaCaT cells （P 〈 0.05）. The cellular ROS, 8-oHdG, and MDA levels significantly increased （P 〈 0.05）, while the T-SOD and GSH-Px levels decreased （P〈 0.05） in a dose-independent manner for both HaCaT and HaCaT-SiRNA-Nrf2 cells. The variances of related indicators for HaCaT- SiRNA-Nrf2 cells were statistically higher than the variances for HaCaT ceils in the same concentration group （P 〈 0.05）. The expression levels of total Nrf-2 protein in low-dose group and the expression levels of nuclear Nrf-2 protein in low- ＆ medium-dose groups were significantly increased in normal HaCaT cells （P 〈 0.05）, then decreased with the increase of exposure concentrations. Total Nrf-2 protein expression levels in HaCaT-SiRNA-Nrf2 cells also showed a similar trend described above, but there was no signific

关 键 词：核因子E2相关因子2 纳米二氧化硅 人永生化表皮细胞 氧化应激 

分 类 号：R329.27[医药卫生—人体解剖和组织胚胎学]