紫外线A辐射对人角质形成细胞的损伤作用

目的：研究紫外线A（UVA）辐射对人角质形成细胞（HaCaT）活力和凋亡的影响及损伤机制。方法：采用不同剂量（1、5、10、20、30、40 J/cm²）UVA照射HaCaT细胞建立急性UVA损伤细胞模型；采用四甲基噻唑蓝（MTT）法及流式细胞术分别检测UVA照射后对细胞活力和凋亡的影响；免疫荧光和Western blot分别检测细胞内γH2AX焦点形成及其蛋白表达水平；原子力显微镜直接观测DNA损伤断裂情况。结果：与对照组相比，5~30 J/cm²范围内，细胞活力随UVA照射剂量的增加而降低（P<0.05），且呈现剂量-效应关系（r=0.982，P=0.009）；UVA照射后20 h细胞凋亡率在10~40 J/cm²明显增加且有一定的量-效关系（r=0.936，P=0.008）。在30和40 J/cm² UVA照射后，免疫荧光也可观察到明显的焦点形成；不同剂量照射后均可检测到磷酸化γH2AX蛋白的表达，在5和10 J/cm² UVA照射时磷酸化γH2AX蛋白表达增强最为明显；原子力显微镜观察到在30和40 J/cm² UVA照射后细胞DNA与对照组相比有明显的断裂，并出现许多断片。结论：UVA可诱导DNA链断裂，引起细胞损伤，从而促进HaCaT细胞凋亡并抑制其存活。

UVA ultraviolet radiation on cellular injury to human keratinocytes

OBJECTIVE: To investigate the mechanisms and effect of ultra violet A (UVA) radiation on vitality and apoptosis of human keratinocytes (HaCaT). METHODS: After different doses of UVA irradiation to keratinocytes, MTT and flow cytometric analysis were used to detect cell survival and apoptosis;immunofluorescence and Western blot were used to detect the formation of intracellular γH2AX focus and its protein levels;and atomic force microscopy was used to detect DNA breakage. RESULTS: UVA irradiation inhibited survival of HaCaT cells in a dose-response manner at the range of 5-30 J/cm² (r=0.982,P=0.009). Early apoptotic cell fraction increased significantly at 20^th hour after UVA irradiation,and had an increasing trend at the range of 10-40 J/cm² (r=0.936,P=0.008) with the rising radiation doses. The 30 and 40 J/cm² doses of UVA induced γH2AX foci formation. After different doses of irradiation,γH2AX expression could be observed,moreover,the expression of γH2AX reached the highest level at 5 J/cm² and 10 J/cm² doses. DNA breaks increased significantly over the control group after irradiation,as detected by atomic force microscopy. CONCLUSION: UVA exposure inhibited HaCaT cell survival,promoted its apoptosis and induced DNA chain break,leading to cell death.