摘要
目的:研究紫外线对人皮肤成纤维细胞的老化损伤及c-fos、c-jun、Sirt1蛋白在UVA诱导损伤后的变化。方法:原代培养人皮肤成纤维细胞,采用MTT法测定第5代细胞经不同强度、不同作用时间UVA照射后人成纤细胞的增殖情况,采用SA-β-Gal染色法观察细胞衰老情况,酶联免疫吸附试验(ELISA)测定细胞内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶活性(GSH-Px)及金属基质蛋白酶-1(MMP-1)含量,免疫组化法观察细胞内c-fos、c-jun的表达,实时定量PCR法检测cfos、c-jun、Sirt1 mRNA的表达,Western blot检测c-fos、c-jun、Sirt1蛋白表达。结果:经5 J/cm^2、10 J/cm^2、20 J/cm^2UVA照射72 h后,人皮肤成纤维细胞的增殖能力减弱,人皮肤成纤维细胞在UVA照射下普遍被诱导成衰老状态,经SA-β-Gal染色后观察到细胞衰老明显,不同强度UVA照射能明显降低细胞内SOD、GSH-Px活性,显著提高MMP-1的含量。免疫组化结果显示c-fos、c-jun表达呈阳性,同时c-fos、c-jun、Sirt1的mRNA和蛋白表达水平均显著升高。结论:UVA能诱导人皮肤成纤维细胞形态改变,这一过程与紫外线强度及作用时间相关,经照射后c-fos、c-jun、Sirt1的表达明显升高,启动了光老化的进程。
Objective:To study the aging damage of ultraviolet radiation on human skin fibroblasts and the changes of c-fos,c-jun and Sirt1 proteins after UVA induced injury. Methods: The primary human skin fibroblasts were cultured,and MTT was used to determine the proliferation of fifth generations of human fibroblast after UVA irradiation.The cell senescence was observed by SA-β-Gal staining,and Enzyme linked immunosorbent assay (ELISA) was used to determine the expression of superoxide dismutase (SOD),glutathione peroxidase activity (GSH-Px) and metal matrix protease-1 (MMP-1) in cells.The immunohistochemical method observed the expression of c-fos and c-jun in cells,and the expression of c-fos,c-jun and Sirt1 mRNA was detected by real-time quantitative PCR.Western blot detected c-fos,c-jun and Sirt1 protein expression. Results: After irradiation of 72 h by 5 J/cm 2,10 J/cm 2 and 20 J/cm 2 UVA,the proliferative ability of human skin fibroblasts was reduced.Human skin fibroblasts were generally induced into an aging state under UVA irradiation.After SA-β-Gal staining,significant cell senescence was observed.After irradiation with different intensities of UVA,SOD and GSH-Px activities in cells were significantly decreased,and MMP-1 expression was significantly increased.Immunohistochemistry showed c-fos and c-jun positive expression,and the mRNA and protein expression levels of c-fos,c-jun and Sirt1 increased significantly. Conclusion: UVA can induce morphological changes of human skin fibroblasts and this process is related to the intensity of ultraviolet radiation and the time of action.The expression of c-fos,c-jun and Sirt1 increased significantly and the photoaging process started after irradiation.
作者
刘金娟
杨宏发
李勇坚
陈艳明
LIU Jin-Juan;YANG Hong-Fa;LI Yong-Jian;CHEN Yan-Ming(Dermatology & STD Department,the Second Hospital of Nanhua University,Hengyang 421001,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2019年第9期1053-1058,共6页
Chinese Journal of Immunology
