窄谱中波紫外线对体外培养人黑素细胞自噬水平的影响

目的 观察窄谱中波紫外线（NB?UVB）照射对黑素细胞自噬水平的影响，探讨NB?UVB治疗白癜风的可能机制。方法 体外培养正常人黑素细胞给予单次0（对照组）、50、100和200 mJ/cm2 NB?UVB照射，照射后24 h收集细胞，单丹酰戊二胺染色法（MDC）检测细胞自噬体变化，免疫印迹法检测自噬信号磷酸化磷酸腺苷蛋白激酶（p?AMPK）、磷酸化哺乳动物雷帕霉素靶蛋白（p?mTOR）、微管相关蛋白轻链3Ⅱ/Ⅰ（LC3Ⅱ/Ⅰ）及P62表达变化，透射电镜观察自噬体与黑素小体超微结构变化。免疫印迹结果采用单因素方差分析进行比较，黑素小体、自噬体与自噬溶酶体数量采用Kruskal?Wallis H检验进行统计分析。结果 MDC染色显示，对照组、50、100和200 mJ/cm2 NB?UVB组自噬体染色阳性率分别为（21.83 ± 3.50）、（23.66 ± 4.12）、（38.08 ± 4.10）、（40.23 ± 1.45）%，100、200 mJ/cm2组显著高于对照组和50 mJ/cm2 NB?UVB组（均P < 0.05）。免疫印迹法显示，与对照组相比，100、200 mJ/cm2组p?AMPK、LC3Ⅱ/Ⅰ表达均显著上调（均P < 0.05），而p?mTOR及P62表达均显著下降（均P < 0.05）。透射电镜结果显示，与对照组自噬体与自噬溶酶体数量（1.88 ± 1.18）相比，100、200 mJ/cm2组（5.12 ± 1.13、5.25 ± 1.04）均显著增多（P < 0.05）。50、100、200 mJ/cm2组黑素小体数量（39.12 ± 9.42、57.38 ± 7.11、59.75 ± 15.15）均较对照组（18.50 ± 4.18）显著增多（均P < 0.05）。结论 NB?UVB照射不仅可以促进黑素小体生成，还可以激活黑素细胞的自噬信号通路，促进自噬体及自噬溶酶体的形成，推测其为白癜风的治疗机制之一。

Effect of narrow-band ultraviolet B radiation on the autophagy of cultured human melanocytes in vitro

Wang Min, Geng Qingwei, Gao Yali, Hua You, Song XiuzuDepartment of Dermatology, Hangzhou Clinical College of Anhui Medical University, Hang Zhou Third Hospital, Hangzhou 310009, ChinaCorresponding author: Song Xiuzu, Email: songxiuzu@sina.com 【Abstract】 Objective To evaluate the effect of narrow-band ultraviolet （NB-UVB） radiation on the autophagy of cultured human melanocytes in vitro, and to explore possible mechanisms underlying the treatment of vitiligo by NB-UVB. Methods In vitro cultured human melanocytes were divided into 4 groups to be irradiated with NB-UVB at different irradiation doses of 0 （control group）, 50, 100 and 200 mJ/cm2 （50-, 100- and 200-mJ/cm2 NB-UVB groups） respectively. After 24-hour treatment, the cells were collected, and monodansylcadaverin （MDC） staining was conducted to detect changes of autophagosomes in melanocytes. Western blot analysis was performed to determine the protein of autophagy signals including phosphorylated AMP-activated protein kinase （p-AMPK）, phosphorylated mammalian target of rapamycin （p-mTOR）, microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ（LC3Ⅱ/Ⅰ） and P62, and transmission electron microscopy to observe ultrastructural changes of autophagosomes and melanosomes in the melanocytes. Statistical analysis was done by using one-way analysis of variance （ANOVA） for the comparison of Western blot results, and by Kruskal-Wallis H test for the comparison of the number of melanosomes, autophagosomes and autolysosomes. Results MDC staining showed that the percentages of autophagosome-positive melanocytes were significantly higher in the 100-, 200-mJ/cm2 NB-UVB groups （38.08% ± 4.10%, 40.23% ± 1.45%, respectively） than in the control group （21.83% ± 3.50%, both P < 0.05） and 50 mJ/cm2 NB-UVB group （23.66% ± 4.12%, both P < 0.05）. As Western blot analysis revealed, the 100-, 200-mJ/cm2 NB-UVB groups showed significantly increased of p-AMPK and LC3Ⅱ/Ⅰ, but significantly decreased of p-mTOR and P62 compared with the control group （all P < 0.05）. Transmission electron microscopy showed that the number of autophagosomes and autolysosomes was significantly higher in the 100-, 200-mJ/cm2 NB-UVB groups （5.12 ± 1.13, 5.25 ± 1.04） than in the control group （1.88 ± 1.18, both P < 0.05 ）. Meanwhile, the number of melanosomes was significantly higher in the 50-, 100- and 200-mJ/cm2 NB-UVB groups （39.12 ± 9.42, 57.38 ± 7.11, 59.75 ± 15.15, all P < 0.05） than in the control group （18.50 ± 4.18, all P < 0.05）. Conclusion NB-UVB radiation can not only promote the formation of melanosomes, but also activate the autophagy signal pathways in the melanocytes and promote the formation of autophagosomes and autolysosomes, which may be one of the mechanisms underlying the treatment of vitiligo by NB-UVB.