人角质细胞紫外线损伤模型筛选抗氧化剂的研究

The screening study of antioxidant to UV-induced HaCat model

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 investigate the influence of antioxidant to the HaCaT cell line which was induced by UV-radiation and to establish a fast screening method. Methods The HaCaT cells were cultured in vitro and then induced oxidation lesion model by UV- radiation, UVA and UVB dose are 5 J/cm2 and 0. 6 J/cm2 respectively. The test groups were replaced with POCI and LBPC antioxidant medium, which dose are highest concentration with no cytotoxicity, and set the negative control, radiation group, Vit C positive control meanwhile. The cells viability, ROS, SOD, cell cycle and cell apoptosis were measured using MTT assay, DCFH-DA labeling, PI fluorescein labeling and Annexinv/PI fluorescein di-labeling method respectively. Results The UV could induce the HaCaT oxidative lesion obviously. The SOD levels in test groups were higher than radiation group clearly（P 〈 0. 001 ）. All ROS fluorescence intensity, apoptosis cell and the percentage of S-phase cell in cell cycle were significantly decrease in test group （ P 〈 0. 001 ）. The antioxidant ability of POCI is better than LBPC, but both lower than Vit C. Conclusion Detecting the protection of antioxidants to HaCaT cell model induced by UV radiation, could provided the predictive to judgment antioxidant properties and strength.