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[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 stress injury induced by nano-SiO_2 in human immortalized epidermal cells with nuclear factor-2 (Nrf-2) deficiency. [Method] The defective cell model of human immortalized epidermal cell (HaCaT) Nrf-2 gene was constructed by using RNA interference technique. The final concentration of nano-SiO (215 nm) Pathological analysis was used to detect the expression of intracellular reactive oxygen species (ROS), 8-oHdG, MDA, total superoxide dismutase (SOD) in HaCaT cells and Nrf- (T-SOD), glutathione peroxidase (GSH-Px) and other oxidative stress indicators, and the total protein and nuclear protein Nrf-2 protein expression levels were analyzed. [Results] 5.0, 10.0 mg / L nano-SiO 2 could significantly decrease the viability of HaCaT cells (P <0.05), while 2.5 mg / L nano-SiO 2 could induce a dose-dependent decrease in the viability of Nrf- (r = -0.914, P <0.05). The viability of Nrf-2-deficient cells was significantly lower than that of HaCaT cells (P <0.05). The levels of ROS, 8-oHdG and MDA in each concentration group increased in a dose-dependent manner and the concentrations of T-SOD and GSH-Px decreased in a dose-dependent manner. Compared with HaCaT cells in the same concentration group, Nrf-2 gene Defective cells in the degree of variation of the relevant indicators greater, the difference was statistically significant (P <0.05). In normal HaCaT cells, the expression levels of Nrf-2 protein and Nrf-2 protein in the low and medium dose groups increased significantly (P <0.05), while with the increased concentration of nano-SiO 2, The expression level also showed a decreasing trend. Compared with the negative control of HaCaT cells, the expression of Nrf-2 protein in Nrf-2-deficient cells was decreased and the expression level of nuclear Nrf-2 protein was not different. [Conclusion] 5.0 mg / L nano-SiO 2 can induce oxidative stress injury in HaCaT cells in vitro. Defective Nrf-2 gene can increase the sensitivity of HaCaT cells to nano-SiO 2 by altering the antioxidant defense ability of HaCaT cells.