8-甲氧补骨脂素和长波紫外线在细胞光老化中对端粒缩短机制的影响

Mechanism of telomere shortening in photoaging model induced by 8-methoxypsoralen and ultraviolet A

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,P21^WAF-1及P16^INK-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,P21^WAF-1及P16^INK-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.

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.