摘要
目的 探讨缝隙连接蛋白43(connexin 43,Cx43)在长波紫外线(ultraviolet A,UVA)诱发人皮肤光老化中的作用。方法 (1)体外培养原代人皮肤成纤维细胞(human dermal fibroblasts, HDFs),分为对照组、UVA照射组,采用CCK-8实验筛选出安全条件后,使用5 J/cm;UVA照射实验组细胞1 h;在照射后0 h、6 h、12 h、24 h分别收集细胞及培养上清液,采用ELISA法检测细胞上清中基质金属蛋白酶-1(matrix metalloproteinases-1,MMP-1)和MMP-9的水平;采用qRT-PCR法和Western blot法检测细胞内Cx43 mRNA和蛋白的表达水平。(2)瞬时转染Cx43-siRNA,采用ELISA法检测细胞上清中MMP-1和MMP-9的水平。(3)将HDFs分为对照组、UVA组、UVA+TNF受体1中和抗体组,经UVA照射后,采用qRT-PCR和ELISA法检测细胞中Cx43 mRNA表达和上清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和MMP-1水平。结果 (1)与对照组相比,UVA组MMP-1和MMP-9水平呈时间依赖性上升(P<0.001),同时Cx43 mRNA和蛋白表达水平均明显下降(P<0.01);(2)Cx43-siRNA显著下调Cx43表达后,MMP-1和MMP-9水平显著上升(P<0.001);(3)与对照组相比,UVA组TNF-α和MMP-1水平显著上升(P<0.01),同时UVA+TNF受体1中和抗体组Cx43和MMP-1水平无明显升高。结论 Cx43可能参与介导了UVA通过诱导TNF-α信号通路引发的皮肤光老化过程,这种介导作用可能是通过Cx43对皮肤中MMP-1的调控得以实现。
Objective To investigate the role of connexin 43(Cx43) in skin photoaging induced by ultraviolet A(UVA).Methods(1) Primary human dermal fibroblasts(HDFs) cultured in vitro, were divided into control group and experiment(UVA-irradiated) group, the HDFs of experiment group were irradiated with UVA(5 J/cm^(2)) for 1 h, after screening out the safe conditions by CCK-8 experiment, then the cells and culture supernatants were collected at 0 h, 6 h, 12 h and 24 h, respectively after irradiated, detect the levels of MMP-1 and MMP-9 in supernatants by ELISA,measure the expression of Cx43 mRNA and the content of protein in HDFs by qRT-PCR and Western blot.(2) After transient transfection with Cx43-siRNA,the levels of MMP-1 and MMP-9 in supernatants were detected by ELISA.After UVA irradiation, the expressions of Cx43 mRNA in the HDFs from control, UVA and UVA+TNF receptor 1 neutralizing antibody groups, and the levels of TNF-α,and MMP-1 in cell supernatant were detected by qRT-PCR and ELISA,respectively.Results The results showed that(1) Compared with control group, the levels of MMP-1 and MMP-9 in UVA group increased in a time-dependent manner(P<0.01),while the expression of mRNA and protein of Cx43 were decreased significantly(P<0.01).(2) The levels of MMP-1 and MMP-9 were significantly increased(P<0.01) after Cx43 expression was down-regulated by Cx43-siRNA.(3) Compared with control group, the levels of TNF-α and MMP-1 in UVA group were significantly increased(P<0.01),meanwhile, the levels of Cx43 and MMP-1 in UVA + TNF receptor 1 neutralizing antibody group were not increased.Conclusion The results suggested that Cx43 may be involved in the photoaging process of skin induced by UVA via the induction of TNF-α signaling pathway, and this mediating effect may be achieved through the regulation of MMP-1 in skin by Cx43.
作者
史正涵
娄炜增
任群翔
黄涛
SHI Zheng-han;LOU Wei-zeng;REN Qun-xiang;HUANG Tao(Shenyang Medical College,Shenyang 110034,China)
出处
《中国工业医学杂志》
CAS
2022年第2期110-114,共5页
Chinese Journal of Industrial Medicine
基金
国家自然科学基金(31400706)
辽宁省科技厅科学技术计划项目(20141098)
辽宁省教育厅科学技术研究项目(SYYX202001)。
