CAJ | 학술논문

目的研究MAPK信号通路调控长波紫外线(UVA)诱导皮肤成纤维细胞组织蛋白酶K(CatK)的表达.方法原代培养的皮肤成纤维细胞取自儿童包皮.Western印迹检测10 J/cm2 UVA照射前及照射后0.75、1.5、3和6h皮肤成纤维细胞中磷酸化JNK(p-JNK)、JNK、磷酸化P38(p-P38)、P38蛋白的表达.800 nmol/L SP600125(SP)、10 μmol/L SB203580(SB)分别孵育皮肤成纤维细胞,实验分为无UVA照射的对照组、SP组、SB组及10 J/cm2 UVA照射的对照(UVA)组、UVA-SP组、UVA-SB组.先以Western印迹检测各组UVA照射后1.5 h磷酸化c-Jun (p-c-Jun)和磷酸化MAPKAPK2 (p-MAPKAPK2)表达,再以RT-PCR和Western印迹检测各组照射后48 h CatK mRNA、蛋白的表达.结果皮肤成纤维细胞在UVA照射后0.75、1.5 h,p-JNK表达的灰度值分别为4.77±0.19和4.68土0.09,p-P38分别为2.44±0.13、2.30±0.04,均较照射前(p-JNK为3.2±0.27,p-P38为1.61±0.08)显著升高(均P＜0.05);而在照射后3、6h的表达与照射前相比差异无统计学意义(P＞0.05).p-c-Jun在UVA-SP组表达(2.55±0.48)、p-MAPKAPK2在UVA-SB组表达(1.16±0.12)均显著低于UVA组(分别为4.85±0.96和2.46±0.09),均P＜0.05.UVA-SP组、UVA-SB组CatK mRNA、蛋白的表达分别降为UVA组的38.9％、28.7％和55.7％、49.6％(P＜0.05),UVA-SP组CatKmRNA、蛋白的表达也均显著低于UVA-SB组(P＜0.05).结论 JNK和P38信号通路在调控UVA上调的皮肤成纤维细胞CatK表达中起重要作用.
목적 연구MAPK신호통로조공장파자외선(UVA)유도피부성섬유세포조직단백매K(CatK)적표체.방법 원대배양적피부성섬유세포취자인동포피.Western인적검측10 J/cm2 UVA조사전급조사후0.75、1.5、3화6h피부성섬유세포중린산화JNK(p-JNK)、JNK、린산화P38(p-P38)、P38단백적표체.800 nmol/L SP600125(SP)、10 μmol/L SB203580(SB)분별부육피부성섬유세포,실험분위무UVA조사적대조조、SP조、SB조급10 J/cm2 UVA조사적대조(UVA)조、UVA-SP조、UVA-SB조.선이Western인적검측각조UVA조사후1.5 h린산화c-Jun (p-c-Jun)화린산화MAPKAPK2 (p-MAPKAPK2)표체,재이RT-PCR화Western인적검측각조조사후48 h CatK mRNA、단백적표체.결과 피부성섬유세포재UVA조사후0.75、1.5 h,p-JNK표체적회도치분별위4.77±0.19화4.68토0.09,p-P38분별위2.44±0.13、2.30±0.04,균교조사전(p-JNK위3.2±0.27,p-P38위1.61±0.08)현저승고(균P＜0.05);이재조사후3、6h적표체여조사전상비차이무통계학의의(P＞0.05).p-c-Jun재UVA-SP조표체(2.55±0.48)、p-MAPKAPK2재UVA-SB조표체(1.16±0.12)균현저저우UVA조(분별위4.85±0.96화2.46±0.09),균P＜0.05.UVA-SP조、UVA-SB조CatK mRNA、단백적표체분별강위UVA조적38.9％、28.7％화55.7％、49.6％(P＜0.05),UVA-SP조CatKmRNA、단백적표체야균현저저우UVA-SB조(P＜0.05).결론 JNK화P38신호통로재조공UVA상조적피부성섬유세포CatK표체중기중요작용.
Objective To investigate whether ultraviolet A UVA)-induced CatK expression is regulated by the mitogen-activated protein kinases (MAPK) signaling pathway in human dermal fibroblasts in vitro.Methods Human dermal fibroblasts were obtained from circumcised foreskin of children,and subjected to primary culture.After several passages of subculture,some fibroblasts were irradiated with UVA at a dose of 10 J/cm2.Western blot was performed to measure the expressions of total and phosphorylated JNK (t-and p-JNK) and P38 (t-and p-P38) at 0.75,1.5,3 and 6 hours after the irradiation.Some fibroblasts were divided into six groups:control group receiving no treatment,SP group treated with SP600125 of 800 nmol/L,SB group treated with SB203580 of 10 μmol/L,UVA group irradiated with UVA at a dose of 10 J/cm2,UVA-SP group treated with SP600125 for 1 hour before and for 1.5 or 48 hours after UVA irradiation at 10 J/cm2,UVA-SB group treated with SB203580 for 1 hour before and for 1.5 or 48 hours after UVA radiation at 10 J/cm2.Subsequently,Western blot was performed to determine the expressions of p-c-Jun and p-MAPKAPK2 in these groups at 1.5 hours after the UVA irradiation,and reverse transcription (RT)-PCR and Western blot to detect the mRNA and protein expressions of CatK at 48 hours after the UVA irradiation,respectively.Statistical analysis was carried out by t test,one way analysis of variance and least significant difference (LSD)-t test.Results The expression levels (gray values) of p-JNK and p-P38 were significantly increased at 0.75 hour (4.77 ± 0.19 and 2.44 ± 0.13 respectively,both P ＜ 0.05) and 1.5 hours (4.68 ± 0.09 and 2.30 ± 0.04 respectively,both P ＜ 0.05),but showed no significant changes at 3 hours (both P ＞ 0.05) and 6 hours (both P ＞ 0.05) after the UVA irradiation compared with those before the irradiation (3.2 ± 0.27 and 1.61 ± 0.08 respectively).A significant decrease was observed in the expression of p-c-Jun in the UVA-SP group and p-MAPKAPK2 in the UVA-SB group compared with the UVA group (p-c-Jun,2.55 ± 0.48 vs.4.85 ±0.96; p-MAPKAPK2,1.16 ± 0.12 vs.2.46 ± 0.09,both P ＜ 0.05).The CatK mRNA and protein expressions were attenuated by 61.1％ and 44.3％ respectively in the UVA-SP group (both P ＜ 0.05),and by 71.3％ and 50.4％ respectively in the UVA-SB group (both P ＜ 0.05) in comparison with the UVA group.The UVA-SP group also showed a significant reduction in CatK mRNA and protein expressions as compared with the UVA-SB group (both P ＜ 0.05).Conclusion Both JNK and P38 signaling pathways,especially the JNK pathway,may contribute to the upregulation of CatK expression in dermal fibroblasts induced by UVA irradiation.