目的 探究他克莫司对白癜风小鼠辅助性T17（Th17）/调节性T （Treg）细胞平衡和黑素细胞丢失的影响。方法 将小鼠随机分为对照组、模型组和他克莫司低、中、高剂量组（涂抹10、50、100 mg 0.03%他克莫司软膏），使用松香/蜡混合物对小鼠背部相同位置进行去毛，大小为2 cm×2 cm，将其分为两部分：用药区域与非用药区域，面积1：1。除对照组外，于小鼠背部用药区均匀涂抹50 mg 40%莫诺苯宗软膏，每天1次，连续90 d，制备白癜风模型。莫诺苯宗涂抹30 d后开始于相同位置涂抹他克莫司软膏，2种药膏涂抹时间间隔7 h，他克莫司软膏每天早晚各涂抹1次，连续用药60 d。肉眼观察小鼠皮毛脱色情况并进行脱色评分，通过反射式共聚焦显微镜（RCM）观察小鼠皮肤黑素细胞和黑色素的分布，取小鼠皮损进行苏木精-伊红染色（HE）和马松染色（MF）对基底层黑素细胞和含黑色素的毛囊进行计数，通过流式细胞术测定小鼠外周血中Th17和Treg淋巴细胞的比例，酶联免疫吸附试验（ELISA）检测外周血白细胞介素-17（IL-17）、IL-22和叉头型基因p3（Foxp3）的含量。结果 与对照组比较，白癜风模型组小鼠用药区皮毛明显脱色，脱色评分显著升高（P<0.05），皮损处色素明显缺失；含黑色素的毛囊和黑素细胞显著减少（P<0.05）；Th17/Treg淋巴细胞比例显著升高（P<0.05）；血清IL-17、IL-22水平显著上升，Foxp3水平显著降低（P<0.05）。与模型组比较，他克莫司各组小鼠皮毛脱色情况明显改善，脱色评分显著降低（P<0.05），皮损处可见色素分布及黑素细胞；含黑色素的毛囊和黑素细胞显著增多（P<0.05）；Th17/Treg淋巴细胞比例显著降低（P<0.05）；IL-17、IL-22水平显著降低，Foxp3水平显著升高（均P<0.05）。结论 他克莫司可调控白癜风小鼠Th17/Treg淋巴细胞平衡，抑制其黑素细胞丢失。

Objective To investigate the effect of tacrolimus on the balance of helper T17 (Th17)/regulatory T (Treg) cells and the loss of melanocytes (MC) in vitiligo mice. Methods The mice were divided into control group, model group and tacrolimus Low， medium and high dose groups (10, 50, 100 mg 0.03% tacrolimus ointment). Rosin/wax mixture was used to remove hair from the same position on the back of mice, with a size of 2 cm×2 cm, which was divided into two parts: drug area and non-drug area, with an area of 1:1. Except for the control group, the vitiligo model was prepared by evenly applying 50 mg 40% Monobenzone ointment on the back of mice, once a day for 60 days. 30 d after monobenzone was applied, tacrolimus ointment was applied at the same position at an interval of 7 h. Tacrolimus ointment was applied once every morning and evening for consecutive 60 d. The decolorization of mouse hair was observed and evaluated by naked eyes, the distribution of melanocytes and melanin in mouse skin was observed by reflection confocal microscopy (RCM), the melanocytes in the basal layer and the hair follicles containing melanin were counted by hematoxylin eosin staining (HE) and Masson's staining (MF), the number of CD4⁺ interleukin (IL)-17⁺ T cells and CD4⁺ CD25⁺ Foxp3⁺ T cells in peripheral blood of mice were measured by flow cytometry, the contents of IL-17, IL-22 and forkhead box P3 (Foxp3) in peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA). Results Comparedwith control group, the skin in test area of mice in the vitiligo model group was significantly decolorized, and the pigmentation of skin lesions was lost, the hair follicles containing melanin and melanocytes decreased significantly, the percentage of Th17/Treg lymphocytes increased significantly, the contents of IL-17 and IL-22 increased significantly, the content of Foxp3 decreased significantly (all P < 0.05). Compared with vitiligo model group, the decolorization of the skin of tacrolimus groups was significantly improved, the distribution of melanin and melanocytes were observed in the lesion, the hair follicles containing melanin and melanocytes increased significantly, the percentage of Th17/Treg lymphocytes decreased significantly, the contents of IL-17 and IL-22 decreased significantly, the content of Foxp3 increased significantly (all P < 0.05). Conclusion Tacrolimus can regulate Th17/Treg lymphocyte balance and inhibit melanocyte loss in vitiligo mice.

R965

陕西省重点研发计划项目（2017SF-153）