目的 探究他克莫司對白癜風小鼠輔助性T17（Th17）/調(diào)節(jié)性T （Treg）細胞平衡和黑素細胞丟失的影響。方法 將小鼠隨機分為對照組、模型組和他克莫司低、中、高劑量組（涂抹10、50、100 mg 0.03%他克莫司軟膏），使用松香/蠟混合物對小鼠背部相同位置進行去毛，大小為2 cm×2 cm，將其分為兩部分：用藥區(qū)域與非用藥區(qū)域，面積1：1。除對照組外，于小鼠背部用藥區(qū)均勻涂抹50 mg 40%莫諾苯宗軟膏，每天1次，連續(xù)90 d，制備白癜風模型。莫諾苯宗涂抹30 d后開始于相同位置涂抹他克莫司軟膏，2種藥膏涂抹時間間隔7 h，他克莫司軟膏每天早晚各涂抹1次，連續(xù)用藥60 d。肉眼觀察小鼠皮毛脫色情況并進行脫色評分，通過反射式共聚焦顯微鏡（RCM）觀察小鼠皮膚黑素細胞和黑色素的分布，取小鼠皮損進行蘇木精-伊紅染色（HE）和馬松染色（MF）對基底層黑素細胞和含黑色素的毛囊進行計數(shù)，通過流式細胞術(shù)測定小鼠外周血中Th17和Treg淋巴細胞的比例，酶聯(lián)免疫吸附試驗（ELISA）檢測外周血白細胞介素-17（IL-17）、IL-22和叉頭型基因p3（Foxp3）的含量。結(jié)果 與對照組比較，白癜風模型組小鼠用藥區(qū)皮毛明顯脫色，脫色評分顯著升高（P<0.05），皮損處色素明顯缺失；含黑色素的毛囊和黑素細胞顯著減少（P<0.05）；Th17/Treg淋巴細胞比例顯著升高（P<0.05）；血清IL-17、IL-22水平顯著上升，F(xiàn)oxp3水平顯著降低（P<0.05）。與模型組比較，他克莫司各組小鼠皮毛脫色情況明顯改善，脫色評分顯著降低（P<0.05），皮損處可見色素分布及黑素細胞；含黑色素的毛囊和黑素細胞顯著增多（P<0.05）；Th17/Treg淋巴細胞比例顯著降低（P<0.05）；IL-17、IL-22水平顯著降低，F(xiàn)oxp3水平顯著升高（均P<0.05）。結(jié)論 他克莫司可調(diào)控白癜風小鼠Th17/Treg淋巴細胞平衡，抑制其黑素細胞丟失。

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

陜西省重點研發(fā)計劃項目（2017SF-153）