摘要
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、210、20、30、40 J/cm）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2测DNA损伤断裂情况。结果：与对照组相比,5-30 J/cm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2应关系（r=0.982,P=0.009）;UVA照射后20 h细胞凋亡率在10-40 J/cm明显增加且有一定的量-效关系（r=0.936,P=0.008）。在30和402 2J/cm 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/cm2UVA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40J/cm 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。
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 yH2AX focus and its protein levels;and atomic force microscopy was used to detect DNA breakage. RE SULTS ：UVA irradiation inhibi ted survival of HaCaT cells in a dose-response manner at the range of 5-30 J/cm^2 （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/cm^2 （r=0.936, P=0.008） with the rising radiation doses. The 30 and 40 J/cm^2 doses of UVA induced yH2AX foci formation. After different doses of irradiation, yH2AX expression could be observed,moreover,the expression of yH2AX reached the highest level at 5 J/cm^2 and 10 J/cm^2 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.
出处
《癌变．畸变．突变》
CAS
CSCD
2016年第5期364-368,共5页
Carcinogenesis,Teratogenesis & Mutagenesis
