中华皮肤科杂志 ›› 2021, Vol. 54 ›› Issue (10): 878-883.doi: 10.35541/cjd.20200894

• 论著 • 上一篇    下一篇

卵巢滤泡激素在干扰素γ介导的黑素细胞凋亡以及趋化因子分泌中的作用研究

周妙妮    林福全    祝逸平    金嵘    盛安琪    许文    许爱娥   

  1. 杭州市第三人民医院皮肤科  310009
  • 收稿日期:2020-09-08 修回日期:2021-08-02 发布日期:2021-09-28
  • 通讯作者: 许爱娥 E-mail:xuaiehz@msn.com
  • 基金资助:
    国家自然科学基金（81803131、81773335）；浙江省自然科学基金（LY21H110002、LY18H110001）；浙江省基础公益研究计划项目（LGF18H110002）；浙江省医药卫生科技计划项目（2019RC072）

Role of folliculin in interferon-γ-mediated apoptosis of and chemokine secretion by melanocytes

Zhou Miaoni, Lin Fuquan, Zhu Yiping, Jin Rong, Sheng Anqi, Xu Wen, Xu Ai′e    

  1. Department of Dermatology, The Third People′s Hospital of Hangzhou, Hangzhou 310009, China
  • Received:2020-09-08 Revised:2021-08-02 Published:2021-09-28
  • Contact: Xu Ai′e E-mail:xuaiehz@msn.com
  • Supported by:
    National Natural Science Foundation of China （81803131, 81773335）; Natural Science Foundation of Zhejiang Province （LY21H110002，LY18H110001）; Basic Public Welfare Research Program of Zhejiang Province （LGF18H110002）; Zhejiang Medicine and Health Technology Program （2019RC072）

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摘要： 【摘要】 目的 研究卵巢滤泡激素（FLCN）在干扰素γ（IFN-γ）介导的黑素细胞凋亡及趋化因子分泌中发挥的作用。方法 从正常人包皮环切组织及白癜风移植患者正常部位的吸疱表皮分离正常原代黑素细胞及白癜风原代黑素细胞。采用Western印迹检测正常原代黑素细胞、白癜风原代黑素细胞及人原代黑素细胞系PIG1细胞中FLCN蛋白的表达。以10 ng/ml IFN-γ刺激48 h的PIG1细胞为诱导组，未经处理的PIG1细胞为对照组，采用qRT-PCR检测两组细胞FLCN及自噬相关微管相关蛋白1轻链3（LC3）Ⅱ、Beclin基因mRNA表达，Western印迹检测FLCN、Beclin1、LC3Ⅱ/Ⅰ水平及腺苷酸激活蛋白激酶（AMPK）、哺乳动物雷帕霉素靶蛋白（mTOR）的磷酸化水平。进一步对采用10 ng/ml IFN-γ刺激的黑素细胞感染FLCN抑制病毒进行不同的处理，分为阴性对照组、FLCN抑制组、抑制FLCN及添加mTOR抑制剂的自噬增强组和抑制FLCN及添加AMPK抑制剂的自噬抑制组。采用流式细胞仪检测各组PIG1细胞凋亡，酶联免疫吸附实验检测各组细胞上清液中趋化因子CXCL10和CCL20的浓度。多组间计量资料比较采用单因素方差分析，组间比较采用LSD-t检验。结果 正常原代黑素细胞（0.850 ± 0.120）、白癜风原代黑素细胞（1.507 ± 0.170）和PIG1细胞（0.697 ± 0.130）FLCN蛋白的相对表达差异有统计学意义（F = 50.09，P < 0.001），白癜风原代黑素细胞高于正常原代黑素细胞和PIG1细胞（t = 4.06、5.89，均P < 0.01）。诱导组FLCN mRNA及蛋白相对表达均高于对照组（均P < 0.01），LC3Ⅱ、Beclin mRNA及蛋白相对表达均低于对照组（均P < 0.01）；诱导组PIG1细胞LC3Ⅱ/Ⅰ水平（0.72 ± 0.02）、AMPK磷酸化水平（0.714 ± 0.023）低于对照组（1.13 ± 0.02、1.176 ± 0.002，t = 7.34、6.67，均P < 0.01），mTOR磷酸化水平（1.051 ± 0.023）高于对照组（0.451 ± 0.016，t = 3.81，P = 0.009）。对照组、诱导组及各处理组PIG1细胞凋亡率及CXCL10、CCL20浓度差异均有统计学意义（均P < 0.001），诱导组高于对照组，FLCN抑制组低于阴性对照组，自噬增强组低于FLCN抑制组，自噬抑制组高于FLCN抑制组（均P < 0.05）。结论 白癜风黑素细胞中高表达FLCN，FLCN表达以及下游信号通路受IFN-γ调控，FLCN可通过调节自噬参与IFN-γ介导的黑素细胞凋亡和趋化因子分泌。

关键词: 黑素细胞, 白癜风, 干扰素γ, 细胞凋亡, 自噬, PIG1细胞, 卵巢滤泡激素

Abstract: 【Abstract】 Objective To investigate the role of folliculin in apoptosis of and chemokine secretion by melanocytes mediated by interferon-γ （IFN-γ）. Methods Normal primary melanocytes were isolated from circumcised foreskin tissues from a healthy male child, and primary vitiliginous melanocytes were isolated from normally pigmented suction-blistered epidermis from patients with vitiligo after suction blister epidermal grafting. Western blot analysis was performed to determine the folliculin protein expression in normal primary melanocytes, primary vitiliginous melanocytes and a human primary melanocyte line PIG1. PIG1 cells stimulated with 10 ng/ml IFN-γ for 48 hours served as induction group, and untreated PIG1 cells served as control group. Real-time quantitative RCR （qRT-PCR） was performed to determine the mRNA expression of folliculin, autophagy-related microtubule-associated protein 1 light chain 3 （LC3）-Ⅱ and Beclin genes, and Western blot analysis to determine the protein expression of folliculin, Beclin1 and LC3Ⅱ/Ⅰ, as well as phosphorylation levels of adenosine monophosphate-activated protein kinase （AMPK） and mammalian target of rapamycin （mTOR） in the above cells. Furthermore, the melanocytes stimulated with 10 ng/ml IFN-γ for 48 hours were divided into several groups: negative control group infected with an empty lentiviral vector, folliculin inhibition group infected with a folliculin-inhibiting lentivirus, autophagy enhancement group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with a mTOR inhibitor, autophagy inhibition group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with an AMPK inhibitor. Then, flow cytometry was conducted to detect apoptosis of PIG1 cells, and enzyme-linked immunosorbent assay to measure the concentration of chemokines CXCL10 and CCL20 in the culture supernatant of PIG1 cells in the above groups. Measurement data were compared among multiple groups by using one-way analysis of variance, and multiple comparisons were carried out by using least significant difference-t test. Results The relative protein expression level of folliculin significantly differed among the normal primary melanocytes （0.850 ± 0.120）, primary vitiliginous melanocytes （1.507 ± 0.170） and PIG1 cells （0.697 ± 0.130; F = 50.09, P < 0.001）, and was significantly higher in the primary vitiliginous melanocytes than in the normal primary melanocytes and PIG1 cells （t = 4.06, 5.89, respectively, both P < 0.01）. Compared with the control group, the induction group showed significantly increased relative mRNA and protein expression levels of folliculin （both P < 0.01）, but significantly decreased relative mRNA and protein expression levels of LC3Ⅱ and Beclin （all P < 0.01）; moreover, the induction group showed significantly decreased LC3Ⅱ/Ⅰ levels （0.72 ± 0.02） and AMPK phosphorylation levels （0.714 ± 0.023） in the PIG1 cells compared with the control group （1.13 ± 0.02, 1.176 ± 0.002, t = 7.34, 6.67, respectively, both P < 0.01）, but significantly increased mTOR phosphorylation levels （1.051 ± 0.023） compared with the control Group （0.451 ± 0.016, t = 3.81, P = 0.009）. There were significant differences in the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 among the control group, induction group and other treatment groups （all P < 0.001）; specifically, the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 were significantly higher in the induction group than in the control group, lower in the folliculin inhibition group than in the negative control group, lower in the autophagy enhancement group than in the folliculin inhibition group, and higher in the autophagy inhibition group than in the folliculin inhibition group （all P < 0.05）. Conclusions Folliculin is highly expressed in vitiliginous melanocytes. Folliculin expression and downstream signaling pathways are regulated by IFN-γ, and folliculin may participate in IFN-γ-mediated melanocyte apoptosis and chemokine secretion via regulating autophagy.

Key words: Melanocytes, Vitiligo, Interferon-gamma, Apoptosis, Autophagy, PIG1 cell, Folliculin