解剖学报 ›› 2021, Vol. 52 ›› Issue (2): 225-230.doi: 10.16098/j.issn.0529-1356.2021.02.010

• 细胞和分子生物学 • 上一篇    下一篇

核因子E2相关因子2对人永生化角质形成细胞氧化损伤的保护作用

毛志蓉1,2 刘芳1,2 杜杰2 邓莉1 高小青1,2*   

  1. 1.西南医科大学医学基础研究中心; 2.西南医科大学解剖学教研室,四川 泸州 646000
  • 收稿日期:2020-04-14 修回日期:2020-08-11 出版日期:2021-04-06 发布日期:2021-04-06
  • 通讯作者: 高小青 E-mail:lygaoxq@163.com
  • 基金资助:
    Nrf2基因修饰对紫外线致人工皮肤氧化应激损伤的作用研究;Nrf2基因修饰抗紫外线致人工皮肤氧化应激损伤的作用机制研究

 Protective effect of nuclear factor E2-related factor 2 on oxidative injury in human immortalized epidermal cells

MAO Zhi-rong1,2 LIU Fang1,2 DU Jie2 DENG Li1 GAO Xiao-qing1,2*   

  1. 1.Preclinical Medicine Research Center; 2.Department of Anatomy, Southwest Medical University, Sichuan Luzhou 646000, China
  • Received:2020-04-14 Revised:2020-08-11 Online:2021-04-06 Published:2021-04-06
  • Contact: GAO Xiao-qing E-mail:lygaoxq@163.com

摘要:

目的 探讨核因子E2相关因子2 (Nrf2)对过氧化氢(H2O2)诱导的人永生化角质形成细胞(HaCaT)氧化损伤的保护作用。  方法 用慢病毒转染方式使HaCaT过表达Nrf2(Nrf2/HaCaT),并用MTT法和抗Ki67免疫荧光染色法检测其增殖活性。实验分为Nrf2/HaCaT组和HaCaT组,每组5个样本。应用200 μmol/L H2O2处理两组细胞24 h以诱导氧化损伤,并用MTT法检测细胞活性,乳酸脱氢酶(LDH)法检测细胞损伤情况,TUNEL染色检测细胞凋亡,ELISA法和比色法检测氧化应激相关指标Nrf2、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、丙二醛(MDA)和活性氧(ROS),以及炎症相关因子白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α、核因子κB(NF-κB)P65的含量。   结果 Nrf2/HaCaT细胞增殖活性显著高于HaCaT(P<0.05)。经H2O2诱导后,Nrf2/HaCaT活性较HaCaT高(P<0.05),LDH释放及凋亡率较HaCaT低(P<0.05),其上清液中的抗氧化物Nrf2、GSH和SOD水平较HaCaT高(P<0.05),而氧化产物ROS、MDA以及炎症因子IL-6、TNF-α和NF-κB P65水平较HaCaT低(P<0.05)。  结论 Nrf2过表达可促进HaCaT增殖及减轻H2O2诱导的细胞氧化和炎性损伤。

关键词: 核因子E2相关因子2, 过表达, 人永生化角质形成细胞, 细胞增殖, 氧化损伤, 慢病毒转染, 酶联免疫吸附测定, 比色法

Abstract:

Objective To investigate the protective effect of nuclear factor E2-related factor 2(Nrf2)on hydrogen peroxide (H2O2)-induced oxidative injury in human immortalized epidermal cells (HaCaT).    Methods  Nrf2 was overexpressed in HaCaT (Nrf2/HaCaT) through lentiviral transfection, then cell proliferative activity was examined by MTT assay and anti-ki67 immunofluorescent staining. The experiment was divided into Nrf2/HaCaT group and HaCaT group, with five samples in each group. After cells were treated with H2O2 of 200 μmol/L for 24 hours to induce oxidative injury, the cell viability, damage and apoptosis were respectively detected by MTT assay, lactate dehydrogenase (LDH) assay and TUNEL staining. Moreover, the content of the related indicators of oxidative stress, including Nrf2, glutathione(GSH), superoxide dismutase(SOD), malondialdehyde(MDA), reactive oxygen species(ROS), and the content of the inflammation associated factor, comprising interleukin(IL)-6, tumor necrosis factor(TNF)-α, nuclear factor kappa-B(NF-κB)P65 were checked by ELISA assay and colorimetry assay.    Results  The proliferative activity of Nrf2/HaCaT was higher than that of HaCaT (P<0.05). When induced by H2O2 for 24 hours, compared with HaCaT, the cell viability increased significantly (P<0.05), and the LDH release and apoptosis rate decreased significantly in Nrf2/HaCaT (P<0.05). The levels of antioxidant Nrf2, GSH, SOD were higher (P<0.05), and the levels of oxidation product ROS, MDA, and inflammatory factor IL-6, TNF-α and NF-κB P65 were lower in supernatant in Nrf2/HaCaT than those in HaCaT (P<0.05).    Conclusion  Nrf2 overexpression could promote HaCaT proliferation and reduce H2O2-induced oxidative and inflammatory injury.

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