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目的探讨利用人角质细胞系建立紫外线损伤模型,研究抗氧化剂对细胞的影响,以建立快速筛检方法。方法体外培养HaCaT细胞,紫外线照射诱发氧化损伤模型,UVA及UVB照射强度分别为5和0.6 J/cm2。测试组分别更换含POCI和LBPC抗氧化剂的培养液,剂量为无细胞毒性的最大浓度,同时设空白对照组、模型组和抗坏血酸阳性对照组。应用MTT(噻唑蓝)比色法、二氯荧光素(DCFH-DA)标记法、PI荧光素标记法和Annexinv/PI双染法,比较细胞活性、活性氧(ROS)、超氧化物歧化酶(SOD)和细胞周期及细胞凋亡等指标的变化。结果紫外线可造成HaCaT细胞明显氧化损伤。测试组SOD水平与照射组相比明显升高(P<0.001);测试组ROS荧光强度、细胞凋亡率及S期细胞比例均显著降低(P<0.001)。POCI抗氧化强度高于LBPC,两者均弱于VC。结论应用紫外线损伤HaCaT细胞模型检测抗氧化剂的保护作用,可为抗氧化特性及强度的预测提供依据。
Objective To explore the use of human keratinocyte cell line to establish a model of UV damage and study the effect of antioxidants on cells to establish a rapid screening method. Methods HaCaT cells were cultured in vitro. The model of oxidative damage was induced by UV irradiation. The intensity of UVA and UVB irradiation were 5 and 0.6 J / cm2, respectively. The test groups were replaced with POCI and LBPC antioxidant medium, the dose of maximum concentration of non-cytotoxic, also set the blank control group, model group and ascorbate positive control group. Cell viability, reactive oxygen species (ROS) and superoxide dismutase (SOD) were measured by MTT assay, DCFH-DA labeling assay, PI fluorescein labeling assay and Annexinv / (SOD) and cell cycle and apoptosis indicators. Results UV can cause significant oxidative damage of HaCaT cells. The level of SOD in the test group was significantly higher than that in the irradiated group (P <0.001). The fluorescence intensity, apoptosis rate and the proportion of S phase cells in the test group were significantly decreased (P <0.001). POCI has higher antioxidant strength than LBPC, both of which are weaker than VC. Conclusion The protective effect of antioxidant on HaCaT cell line with ultraviolet light damage can provide a basis for the prediction of antioxidant properties and intensity.