中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (5): 375-378.doi: 10.3760/cma.j.issn.0412-4030.2018.05.013

• 研究报道 • 上一篇    下一篇

他克莫司对γ干扰素刺激HaCaT细胞分泌趋化因子CXCL9和CXCL10的影响及机制研究

杨赛琳1,许文2,林福全2,周妙妮3,许爱娥4   

  1. 1. 浙江中医药大学附属杭州第三医院皮肤科
    2. 杭州市第三人民医院
    3. 杭州市第三人民医院皮肤科
    4. 安徽医科大学附属杭州市第三人民医院皮肤科
  • 收稿日期:2017-11-01 修回日期:2018-01-30 出版日期:2018-05-15 发布日期:2018-05-02
  • 通讯作者: 许爱娥 E-mail:xuaiehz@msn.com
  • 基金资助:
    国家自然科学基金;国家自然科学基金;国家自然科学基金;浙江省基础公益研究计划项目;浙江省自然科学基金;浙江省自然科学基金

Effects of tacrolimus on the secretion of chemokines CXCL9 and CXCL10 by γ-interferon-simulated HaCaT cells

  • Received:2017-11-01 Revised:2018-01-30 Online:2018-05-15 Published:2018-05-02
  • Supported by:
    National Natural Science Foundation of China;National Natural Science Foundation of China;National Natural Science Foundation of China;Basic Public Welfare Research Project of Zhejiang Province;Natural Science Foundation of Zhejiang Province of China;Natural Science Foundation of Zhejiang Province of China

摘要: 目的 分析他克莫司对γ干扰素(IFN?γ)刺激下HaCaT细胞分泌CXCL9、CXCL10、p?JAK1、p?STAT1的影响,探讨他克莫司治疗白癜风的作用机制。方法 1、10、20、40、60、80、100、120 mg/L他克莫司预处理HaCaT细胞4 h后, 噻唑蓝法(MTT)检测细胞增殖。HaCaT细胞分为空白对照组, IFN?γ组(500 U/ml),他克莫司组(20 mg/L)及他克莫司 + IFN?γ组。他克莫司预处理HaCaT细胞4 h, IFN?γ刺激细胞12 h或48 h,实时荧光定量PCR检测细胞CXCL9、CXCL10 mRNA表达。Western 印迹法检测细胞CXCL9、CXCL10、p?JAK1、p?STAT1蛋白表达。ELISA法检测细胞培养上清液CXCL9、CXCL10蛋白含量。结果 他克莫司对HaCaT细胞增殖无影响的最大浓度为20 mg/L(P>0.05)。20 mg/L他克莫司预处理HaCaT细胞后,使IFN?γ刺激下HaCaT细胞表达CXCL9、CXCL10 mRNA分别由10 369.08 ± 7.99、290.02 ± 2.16降低至5 914.33 ± 4.59、114.96 ± 0.73(均P<0.01)。CXCL9、CXCL10、p?JAK1、p?STAT1蛋白表达分别由8.47 ± 0.29、7.87 ± 0.17、4.20 ± 0.18、4.29 ± 0.11降低至7.36 ± 0.13、7.36 ± 0.09、2.60 ± 0.16、3.62 ± 0.19(均P<0.01)。细胞培养上清液中CXCL9、CXCL10蛋白含量分别由1 213.36 ± 0.95、1 722.41 ± 2.57降低至426.45 ± 0.31、554.12 ± 0.56(均P<0.01)。结论 他克莫司对IFN?γ刺激下HaCaT细胞分泌CXCL9、CXCL10、p?JAK1、p?STAT1具有抑制作用。

关键词: 白癜风, 干扰素γ, 趋化因子CXCL9, 趋化因子CXCL10, Janus激酶类, STAT1转录因子, HaCaT细胞, 他克莫司

Abstract: Yang Sailin, Xu Wen, Lin Fuquan, Zhou Miaoni, Xu Ai′e Department of Dermatology, Third Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310009, Zhejiang, China Corresponding author: Xu Ai′e, Email: xuaiehz@msn.com 【Abstract】 Objective To analyze effects of tacrolimus on the secretion of chemokines CXCL9 and CXCL10 by γ-interferon (IFN-γ)-simulated HaCaT cells, as well as phosphorylated Janus kinase 1 (p-JAK1) and phosphorylated signal transducer and activator of transcription 1 (p-STAT1), and to explore the mechanism of tacrolimus in the treatment of vitiligo. Methods HaCaT cells were treated with 1, 10, 20, 40, 60, 80, 100, 120 mg/L tacrolimus solution separately for 4 hours, and methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the cellular proliferative activity. HaCaT cells were divided into 4 groups: blank control group receiving no treatment, IFN-γ group treated with 500 U/ml IFN-γ for 12 or 48 hours, tacrolimus group treated with 20 mg/L tacrolimus for 4 hours, and tacrolimus + IFN-γ group treated with 20 mg/L tacrolimus for 4 hours followed by the treatment with 500 U/ml IFN-γ for 12 or 48 hours. Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA of CXCL9 and CXCL10, Western blot analysis to determine the protein of CXCL9, CXCL10, p-JAK1, and p-STAT1, and enzyme-linked immunosorbent assay (ELISA) to detect the levels of CXCL9 and CXCL10 in the culture supernatants of HaCaT cells. Results Tacrolimus at the maximum concentration of 20 mg/L had no effect on the proliferation of HaCaT cells (P > 0.05). After the pretreatment with 20 mg/L tacrolimus, the mRNA of CXCL9 and CXCL10 significantly decreased from 10 369.08 ± 7.99 and 290.02 ± 2.16 to 5 914.33 ± 4.59 and 114.96 ± 0.73, respectively, after the treatment with IFN-γ (both P < 0.01), and the protein of CXCL9, CXCL10, p-JAK1, and p-STAT1 also significantly decreased from 8.47 ± 0.29, 7.87 ± 0.17, 4.20 ± 0.18 and 4.29 ± 0.11 to 7.36 ± 0.13, 7.36 ± 0.09, 2.60 ± 0.16 and 3.62 ± 0.19, respectively, after the treatment with IFN-γ (all P < 0.01). Moreover, the levels of CXCL9 and CXCL10 in the culture supernatants of HaCaT cells significantly decreased in the IFN-γ group (1 213.36 ± 0.95, 1 722.41 ± 2.57, respectively) compared with the tacrolimus + IFN-γ group (426.45 ± 0.31, 554.12 ± 0.56, respectively, both P < 0.01). Conclusion Tacrolimus can inhibit the secretion of CXCL9, CXCL10, p-JAK1 and p-STAT1 by HaCaT cells stimulated by IFN-γ.

Key words: Vitiligo, Interferon?gamma, Chemokine CXCL9, Chemokine CXCL10, Janus kinases, STAT1 transcription factor, HaCaT cells, Tacrolimus