Nrf2蛋白在中波紫外线照射HaCaT细胞中的光保护作用  

Protective effect of Nrf2 protein against ultraviolet B-induced photodamage to HaCaT cells

作　　者：林芳 马良娟[1] 张晓卉[1] 柏冰雪[1] Lin Fang;Ma Liangjuan;Zhang Xiaohui;Bai Bingxue(Department of Dermatology,The 2nd Affiliated Hospital of Harbin Medical University,Harbin 150000,China;Department of Dermatology,Cangzhou Central Hospital,Cangzhou 061000,Hebei,China)

机构地区：[1]哈尔滨医科大学附属第二医院皮肤科,150000 [2]沧州市中心医院皮肤科,河北061000

出　　处：《中华皮肤科杂志》2020年第2期128-132,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81502706)。

摘　　要：目的探讨Nrf2对中波紫外线(UVB)致HaCaT细胞光损伤的保护作用及其作用机制。方法将培养的HaCaT细胞分为对照组、UVB组、Nrf2组、Nrf2+UVB组,Nrf2组、Nrf2+UVB细胞感染Nrf2基因过表达慢病毒,UVB组、Nrf2+UVB组细胞以30 mJ/cm^2的UVB照射30 s,继续培养24 h,观察UVB照射后HaCaT细胞形态的变化,Western印迹检测各组Nrf2蛋白水平,CCK8法检测各组细胞生存率,流式细胞仪检测各组细胞活性氧(ROS)水平,生化法检测超氧化物歧化酶(SOD)水平。多组间均数比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果对照组HaCaT细胞形态为多角形、呈簇集状生长,UVB照射后细胞出现皱缩、变圆.漂浮细胞数增多,贴壁数量明显减少。对照组、UVB组、Nrf2组、Nrf2+UVB组Nrf2蛋白相对表达水平分别为1.84±0.047,0.63±0.082、2.19±0.168、1.43±0.069,差异有统计学意义(F=64.81,P<0.05),Nrf2组水平高于对照组(t=14.82,P<0.05);4组细胞生存率分别为98.00%±2.39%,24.40%±2.98%、71.63%±3.39%、43.38%±3.39%,差异有统计学意义(F=236.66,P<0.05),UVB组细胞活力低于对照组(t=33.34,P<0.05)和Nrf2+UVB组(t=10.07,P<0.05);4组细胞的相对ROS含量分别为1.27±0.10.5.65±0.19,2.10±0.73,3.67±0.19,差异有统计学意义(F=481.39,P<0.05),UVB组高于对照组(t=33.68,P<0.05)和Nrf2+UVB(t=12.47,P<0.05)。4组细胞SOD水平差异有统计学意义(F=170.76,P<0.05),UVB组低于对照组(t=11.25,P<0.05)和Nrf2+UVB组(t=17.52,P<0.05)。4组细胞IL-6水平差异有统计学意义(F=532.34,P<0.05),UVB组高于对照组(t=28.48,P<0.05),Nrf2+UVB组低于UVB组(t=27.82,P<0.05)。4组细胞TNF-α水平差异无统计学意义(F=2.02,P=0.19)。结论Nrf2可以通过降低细胞内ROS水平,提高内源性抗氧化酶SOD的活性,保护细胞免受UVB照射引起的氧化损伤。Objective To evaluate the protective effect of nuclear factor E2-related factor 2(Nrf2)protein against ultraviolet B(UVB)-induced photodamage to HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 4 groups:control group receiving no treatment,UVB group irradiated with 30 mJ/cm^2 UVB for 30 s,Nrf2 group transfected with a lentiviral vector overexpressing the Nrf2 gene,and Nrf2+UVB group transfected with a lentiviral vector overexpressing the Nrf2 gene followed by radiation with 30 mJ/cm^2 UVB for 30 s.After the treatment,HaCaT cells in the above 4 groups were cultured for another 24 hours.Then,changes in the morphology of HaCaT cells were observed after UVB radiation,Western blot analysis was performed to determine Nrf2 protein expression,cell counting kit-8(CCK8)assay to detect survival rates of HaCaT cells,flow cytometry to detect levels of reactive oxygen species(ROS),and a biochemical method to detect superoxide dismutase(SOD)levels in cells,and enzyme-linked immunosorbent assay to detect levels of interleukin(IL)-6 and tumor necrosis factor(TNF)-α in the culture supernatant of HaCaT cells.One-way analysis of variance was used for comparing means in several groups,and least significant difference(LSD)-t test for multiple comparisons.Results Polygonal and clustered HaCaT cells were observed in the control group.After UVB radiation,HaCaT cells became shrunken and round,the number of floating cells increased,and the number of adherent cells markedly decreased.There was a significant difference in Nrf2 protein expression among the control group,UVB group,Nrf2 group and Nrf2+UVB group(1.84±0.047,0.63±0.082,2.19±0.168 and 1.43±0.069 respectively;F=64.81,P<0.05),and the Nrf2 protein expression was significantly higher in the Nrf2 group than in the control group(t=14.82,P<0.05);the survival rates of HaCaT cells also significantly differed among the above 4 groups(98.00%±2.39%,24.40%±2.98%,71.63%±3.39%and 43.38%±3.39% respectively;F=236.66,P<0.05),and the UVB group showed s

关 键 词：NF-E2相关因子2 紫外线 活性氧 超氧化物歧化酶 HACAT细胞 氧化损伤 

分 类 号：R[医药卫生]