CAJ | 학술논문

目的探讨8-甲氧补骨脂素(8-MOP)和长波紫外线(UVA)对人真皮成纤维细胞光老化模型中端粒缩短机制的影响.方法研究对象分为对照组、8-MOP组、UVA组及8-MOP+UVA组,对上述各组采用流式细胞仪检测细胞周期G1期阻滞率、酶组织化学染色法检测老化相关β-半乳糖苷酶(SA-β-Gal)、免疫荧光检测光产物8-羟基脱氧鸟嘌呤(8-oxo-dG)、实时定量PCR检测端粒相对长度,以及用Western印迹检测老化相关蛋白P53,P21WAF-1及P16INK-4a表达水平.结果 8-MOP+UVA组在照光后24、48、72 h及7 d时的G1期阻滞率均高于对照组(61.4%±1.5%比32.8%±1.5%、69.5%±2.2%比44.9%4±2.3%、88.2%±1.6%比59.8%±1.4%、90.7%±2.5%比68.5%±2.6%,均P＜0.01);8-MOP+UVA组在照光后24、48、72 h及7d时的SA-β-Gal阳性细胞比率均明显高于对照组(34.87%±0.59%比7.11%±0.78%、59.38%±0.46%比10.57%±0.47%、72.46%±0.98%比11.67%±0.87%、94.33%±0.13%比12.04%±0.12%,均P＜0.01);8-MOP+UVA组照光后即刻产生的8-oxo-dG的水平(阳性细胞核百分数:95.78%±0.14%)也高于对照组(7.69%±0.09%,P＜0.01)、8-MOP组(9.76%±0.11%,P＜0.01)和UVA组(35.29%±0.14%,P＜0.05);8-MOP+UVA组在照光后7 d时端粒相对长度数值明显低于对照组(2.57±0.05比6.63 ±0.12,P＜0.01),而该组老化相关蛋白P53,P21WAF-1及P16INK-4a水平明显高于对照组(3.00±0.88比0.54±0.10、2.50±0.51比0.42±0.06、2.21±0.34比0.38 ±0.05,均P＜0.01).结论 8-MOP和UVA可通过对端粒基因的氧化应激损伤而加快端粒缩短,影响下游老化相关蛋白表达水平,最终加速细胞老化进程.
목적 탐토8-갑양보골지소(8-MOP)화장파자외선(UVA)대인진피성섬유세포광노화모형중단립축단궤제적영향.방법 연구대상분위대조조、8-MOP조、UVA조급8-MOP+UVA조,대상술각조채용류식세포의검측세포주기G1기조체솔、매조직화학염색법검측노화상관β-반유당감매(SA-β-Gal)、면역형광검측광산물8-간기탈양조표령(8-oxo-dG)、실시정량PCR검측단립상대장도,이급용Western인적검측노화상관단백P53,P21WAF-1급P16INK-4a표체수평.결과 8-MOP+UVA조재조광후24、48、72 h급7 d시적G1기조체솔균고우대조조(61.4%±1.5%비32.8%±1.5%、69.5%±2.2%비44.9%4±2.3%、88.2%±1.6%비59.8%±1.4%、90.7%±2.5%비68.5%±2.6%,균P＜0.01);8-MOP+UVA조재조광후24、48、72 h급7d시적SA-β-Gal양성세포비솔균명현고우대조조(34.87%±0.59%비7.11%±0.78%、59.38%±0.46%비10.57%±0.47%、72.46%±0.98%비11.67%±0.87%、94.33%±0.13%비12.04%±0.12%,균P＜0.01);8-MOP+UVA조조광후즉각산생적8-oxo-dG적수평(양성세포핵백분수:95.78%±0.14%)야고우대조조(7.69%±0.09%,P＜0.01)、8-MOP조(9.76%±0.11%,P＜0.01)화UVA조(35.29%±0.14%,P＜0.05);8-MOP+UVA조재조광후7 d시단립상대장도수치명현저우대조조(2.57±0.05비6.63 ±0.12,P＜0.01),이해조노화상관단백P53,P21WAF-1급P16INK-4a수평명현고우대조조(3.00±0.88비0.54±0.10、2.50±0.51비0.42±0.06、2.21±0.34비0.38 ±0.05,균P＜0.01).결론 8-MOP화UVA가통과대단립기인적양화응격손상이가쾌단립축단,영향하유노화상관단백표체수평,최종가속세포노화진정.
Objective To investigate the mechanism of telomere shortening through 8-methoxypsoralen(8-MOP)and subsequent ultraviolet A(UVA)irradiation-induced photoaging model in human dermal fibroblasts(HDFs).Methotis Photoaging model was established by 8-MOP+UVA in skin HDFs.Flow cytometer.enzyme eytochemistry,immunofluorescence,Westem blot and Real-time PCR were employed.Results The percentage of G1 blockage of 8-MOP+UVA group were higher than that of control group at 24、48、72 h and 7 d(61.4%±1.5% vs 32.8%±1.5%.69.5%±2.2% vs 44.9% ±2.3%.88.2%±1.6% vs 59.8%±1.4%,90.7%±2.5% vs 68.5%±2.6%.all P＜0.01).The expression of SA-β-Gal of 8-MOP+UVA group were higher than that of control group at 24、48、72 h and 7 d(34.87%±0.59% vs 7.11%±0.78%,59.38%±0.46% vs 10.57%±0.47%.72.46%±0.98% vs 11.67%±0.87%,94.33%±0.13% vs 12.04%±0.12%,all P＜0.01).8-MOP+UVA treatment could significantly aggravate the oxidative DNA damages,the percentage of 8-oxo-dG positive cell of 8-MOP+UVA group(95.78%±0.14%)were significantly higher than that of control group(7.69%±0.09%,P＜0.01),8-MOP group(9.76%±0.11%,P＜0.01)and UVA group(35.29%±0.14%,P＜0.05).8-MOP+UVA treatment could accelerate the telomere shortening.the relative length of telomere of 8-MOP +UVA group were 2.57±0.05 lower than that of control group(6.63±0.12.P＜0.01).The levels of P53,P21WAF-1 and P16INK-4a of 8-MOP+UVA group were higher than that of control group(3.00±0.88 vs 0.54±0.10,2.50±0.51 vs 0.42±0.06,2.21±0.34 vs 0.38±0.05,all P＜0.01).Conclusion 8-MOP+UVA-induced photoaging of HDFs can be mediated though the regulation of telomere and subsequent P53-dependent signaling pathways.