UVB诱导人皮肤成纤维细胞的氧化应激损伤研究

目的 观察人皮肤成纤维细胞被UVB照射后的光损伤、凋亡、周期阻滞和氧化应激损伤状态，并检测氧化应激的相关信号蛋白p66Shc的表达情况。方法 用小剂量UVB多次照射培养的人皮肤成纤维细胞，以细胞衰老β-半乳糖苷酶（SA-β-Gal）化学染色法观察细胞衰老状态，流式细胞仪检测细胞凋亡和细胞周期，ELISA法检测细胞内超氧化物歧化酶活性和丙二醛的含量，并以Western印迹法检测p66Shc蛋白的表达。结果 SA-β-Gal染色显示，培养的人皮肤成纤维细胞在UVB照射后呈强阳性染色；细胞凋亡率在未照射的对照组为0.96％，UVB照射组为37％；而细胞周期检测显示，经UVB照射的人皮肤成纤维细胞大部分阻滞于G0/G1期（80.07%）。细胞内超氧化物歧化酶水平对照组为（52.35 ± 4.97） ng/g蛋白，UVB照射组为（7.81 ± 0.68） ng/g蛋白（两组比较，P < 0.01）；而丙二醛水平两组分别为（3.52 ± 0.34） ng/g蛋白和（33.91 ± 3.20） ng/g蛋白（两组比较，P < 0.05）。人皮肤成纤维细胞经UVB照射诱导后24 h，p66Shc呈弱阳性表达，48 h后表达进一步增强。结论 培养的人皮肤成纤维细胞经UVB照射后进入氧化应激增强状态。p66Shc蛋白在此过程中表达逐渐增强。

Oxidative stress in human skin fibroblasts induced by UVB irradiation

Objective To observe the aging,apoptosis,cell cycle arrest and oxidative stress in human skin fibroblast(HSF)induced by UVB,and to detect the expression profiles of p66Shc,a determinant of oxidative stress response and life span,in this process.Methods HSF cells were exposed to UVB at a subcytotoxic dosage twice a day for three days.The cells without exposure served as control.After another 24-hour culture,SA-β-Gal staining was performed to evaluate the senescence state of the cells,flow cytometry to observe cell apoptosis;cell cycle arrest was detected by serum starvation and flow cytometry:ELISA was applied to detect intracellular levels of superoxide dismutase(SOD)and malondialdehvde(MDA),and Western blotting to analyze the expression of p66Shc protein.Results The percentage of cells positive for SA-β-Gal staining increased from 0 to 98.3% after UVB radiation,which strongly suggested an aging state of HSF cells.The percentage of apoptotic cells increased from 0.96% to 37%.and 80.07% of the HSF cells were arrested in G0/G1 phase following the irradiation.Intracellular SOD activity decreased from(52.35±4.97)ng/g to(7.81±0.68)ng/g(P＜0.01).while intracellular MDA was found to increase from(3.52±0.34)ng/g to(33.91±3.20)ng/g(P＜0.05).The p66Shc protein was found to be weakly expressed in HSF in 24 hours following the exposure to UVB,and a stronger expression was noted 48 hours later.Conclusions HSF cells are induced into a state of senescence associated with oxidative stress after UVB irradiation,which may be applied as an in vitro model in aging research.The expression of p66Shc is increased in HSF during this process,and further studies are needed to explore the relation between p66Shc and oxidative stress as well as cellular aging.