中华皮肤科杂志 ›› 2025, Vol. 58 ›› Issue (11): 1042-1052.doi: 10.35541/cjd.20240655

• 论著 • 上一篇    下一篇

银屑病中EPHA2通过ERK通路调控角质形成细胞增殖分化的机制研究

蒋星宇1，2，3    余增洋3，4    马蕊2，3    时熔灿1，2，3    黄大炜2，3    王媛媛2，3    蔡江鲁伊2，3    史玉玲1，2，3   

  1. 1安徽医科大学上海皮肤病临床学院  安徽医科大学第五临床医学院（上海市皮肤病医院），上海  200443；2同济大学附属皮肤病医院皮肤科，上海  200443；3同济大学医学院银屑病研究所，上海  200443；4同济大学附属第十人民医院皮肤科，上海  200072
  • 收稿日期:2024-11-29 修回日期:2025-10-13 发布日期:2025-11-03
  • 通讯作者: 史玉玲 E-mail:shiyuling1973@tongji.edu.cn
  • 基金资助:
    国家自然科学基金（82073429、82273510）；上海市教育委员会科研创新计划项目（2019-01-07-00-07-E00046）；上海申康医院发展中心临床研究项目（SHDC2020CR1014B）

Investigations into the mechanisms underlying the regulatory effect of EPHA2 on keratinocyte proliferation and differentiation via ERK pathway in psoriasis

Jiang Xingyu1,2,3, Yu Zengyang3,4, Ma Rui2,3, Shi Rongcan1,2,3, Huang Dawei2,3, Wang Yuanyuan2,3, Cai Jiangluyi2,3, Shi Yuling1,2,3   

  1. 1Shanghai Skin Disease Clinical College, the Fifth School of Clinical Medicine, Anhui Medical University（Shanghai Skin Disease Hospital）, Shanghai 200443, China; 2Department of Dermatology, Skin Disease Hospital, Tongji University, Shanghai 200443, China; 3Institute of Psoriasis, Tongji University School of Medicine, Shanghai 200443, China; 4Department of Dermatology, Tenth People's Hospital, Tongji University, Shanghai 200072, China
  • Received:2024-11-29 Revised:2025-10-13 Published:2025-11-03
  • Contact: Shi Yuling E-mail:shiyuling1973@tongji.edu.cn
  • Supported by:
    National Natural Science Foundation of China （82073429, 82273510）; Innovation Program of Shanghai Municipal Education Commission （2019-01-07-00-07-E00046）; Clinical Research Plan of Shanghai Hospital Development Center （SHDC2020CR1014B）

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摘要： 【摘要】 目的 探究银屑病皮损中肝配蛋白A型受体2（EPHA2）的表达及其对人表皮角质形成细胞（NHEK）增殖分化的影响。方法 通过基因表达综合（GEO）数据库GDS4602数据集分析银屑病患者皮损中EPHA2基因的表达变化。收集银屑病患者和健康对照各3例，免疫荧光检测皮肤组织中EPHA2的表达。12只BALB/c雌鼠随机分为正常对照组（不接受任何处理）、咪喹莫特组（外用5%咪喹莫特乳膏62.5 mg）、咪喹莫特 + ALWⅡ-41-27组（外用5%咪喹莫特乳膏62.5 mg后，腹腔注射EPHA2抑制剂ALWⅡ-41-27 20 mg·kg-1·d-1），每组4只；连续给药6 d后，取小鼠背部皮肤组织，苏木精-伊红（HE）染色观察病理变化，免疫荧光检测EPHA2及磷酸化的胞外信号调节激酶（p-ERK）1/2蛋白的表达，实时荧光定量PCR（qPCR）检测核增殖抗原（Ki67）、内披蛋白（Ivl）、兜甲蛋白（Lor）、角蛋白10（Krt10）mRNA的表达。体外培养NHEK，分为对照组（不接受任何处理）、M5组（10 ng/ml M5处理）、ALWⅡ-41-27组（1 μmol/L ALWⅡ-41-27处理）和M5 + ALWⅡ-41-27组（10 ng/ml M5 + 1 μmol/L ALWⅡ-41-27同时处理）；刺激24 h后，5-乙炔基-2′-脱氧尿苷（EdU）染色检测细胞增殖活力，Western印迹法检测EPHA2、ERK及其磷酸化的表达，qPCR检测KI67、IVL、LOR、KRT10 mRNA的表达。多组间比较采用单因素方差分析，组间两两比较采用Dunnett′s T3检验，两独立样本组间比较采用独立样本t检验，两配对样本组间比较采用配对t检验。结果 GEO数据库分析显示，与健康对照相比，银屑病患者皮损中EPHA2基因表达上调（t = 21.07，P < 0.001）。免疫荧光检测显示，银屑病患者皮损和银屑病模型小鼠皮损中EPHA2的表达分别高于健康对照正常皮肤和正常对照小鼠皮肤组织（均P < 0.01）。动物实验表明，与正常对照组和咪喹莫特 + ALWⅡ-41-27组相比，咪喹莫特组表皮较厚，Ki67 mRNA表达较高，Ivl、Lor、Krt10 mRNA表达较低，p-ERK1/2蛋白表达较高（均P < 0.05）。细胞实验表明，与对照组和M5 + ALWⅡ-41-27组相比，M5组EdU阳性细胞比例较高（35.61% ± 1.18%比24.83% ± 0.60%、12.49% ± 1.52%，t = 8.12、12.00，P = 0.015、0.001），KI67 mRNA表达较高，IVL、LOR、KRT10 mRNA表达较低（均P < 0.05）；Western印迹检测显示，M5组NHEK中EPHA2及其磷酸化蛋白和p-ERK1/2蛋白表达量高于对照组和M5 + ALWⅡ-41-27组（均P < 0.05），各组间ERK1/2蛋白表达量的差异无统计学意义（P > 0.05）。结论 EPHA2在银屑病皮损中表达升高，可能通过ERK通路促进角质形成细胞的增殖并抑制其分化。

关键词: 银屑病, 肝配蛋白A型受体2, 角质形成细胞, 细胞增殖, 细胞分化, 胞外信号调节激酶通路

Abstract: 【Abstract】 Objective To investigate the expression of ephrin type-A receptor 2 （EPHA2） in psoriatic lesions and its effect on the proliferation and differentiation of normal human epidermal keratinocytes （NHEKs）. Methods The GDS4602 dataset from the Gene Expression Omnibus （GEO） database was analyzed to determine EPHA2 gene expression changes in psoriatic lesions. Skin tissue samples were collected from 3 psoriasis patients and 3 healthy controls, and EPHA2 expression was determined in the skin tissues by immunofluorescence staining. Twelve female BALB/c mice were randomly divided into 3 groups （4 mice in each group）: a normal control group （receiving no treatment）, an imiquimod group （topically treated with 62.5 mg of imiquimod 5% cream）, and an imiquimod + ALWⅡ-41-27 group （topically treated with 62.5 mg of imiquimod 5% cream, followed by intraperitoneal injections of the EPHA2 inhibitor ALWⅡ-41-27 at a dose of 20 mg·kg?1·d?1）; after 6 days of treatment, dorsal skin samples were harvested for hematoxylin-eosin （HE） staining, immunofluorescence staining was performed to determine the expression of EPHA2 and phosphorylated extracellular signal-regulated kinase 1/2 （p-ERK1/2）, and real-time fluorescence-based quantitative PCR （qPCR） was conducted to determine the mRNA expression of the nuclear proliferation antigen Ki67, involucrin （Ivl）, loricrin （Lor）, and keratin 10 （Krt10）. In vitro cultured NHEKs were divided into a control group （receiving no treatment）, an M5 group （treated with 10 ng/ml M5 cytokines ［including interleukin-17A, interleukin-22, interleukin-1α, oncostatin M and tumor necrosis factor-α］）, an ALWⅡ-41-27 group （treated with 1 μmol/L ALWⅡ-41-27）, and an M5 + ALWⅡ-41-27 group （treated with 10 ng/ml M5 and 1 μmol/L ALWⅡ-41-27）; after 24 hours of treatment, the 5-ethynyl-2'-deoxyuridine （EdU） assay was performed to assess cellular proliferative activity, Western blot analysis to determine the expression of EPHA2, ERK and their phosphorylated proteins, and qPCR to determine the mRNA expression of KI67, IVL, LOR, and KRT10. One-way analysis of variance, Dunnett's T3 test, two-independent-sample t test, and paired t test were used for statistical analysis. Results GEO database analysis revealed upregulated EPHA2 expression in psoriatic lesions compared with normal skin tissues from healthy controls （t = 21.07, P < 0.001）. Immunofluorescence staining showed increased EPHA2 expression in skin tissues from psoriasis patients and mouse models of psoriasis compared with those from healthy controls and normal control mice, respectively （both P < 0.01）. In the animal experiments, the imiquimod group showed thicker epidermis, increased Ki67 mRNA expression, decreased mRNA expression of Ivl, Lor, and Krt10, and elevated p-ERK1/2 expression compared with the normal control group and imiquimod + ALWⅡ-41-27 group （all P < 0.05）. In the cell experiments, the M5 group showed an increased proportion of EdU-positive cells （35.61% ± 1.18% vs. 24.83% ± 0.60% and 12.49% ± 1.52%, t = 8.12, 12.00, P = 0.015, 0.001, respectively）, increased KI67 mRNA expression, and decreased mRNA expression of IVL, LOR, and KRT10 compared with the control group and M5 + ALWⅡ-41-27 group （all P < 0.05）; Western blot analysis revealed that the expression levels of EPHA2, p-EPHA2, and p-ERK1/2 in NHEKs were significantly higher in the M5 group than in the control group and M5 + ALWⅡ-41-27 group （all P < 0.05）, but there was no significant difference in the ERK1/2 protein expression among groups （P > 0.05）. Conclusion EPHA2 expression was upregulated in psoriatic lesions, which may promote keratinocyte proliferation and inhibit its differentiation, possibly via the ERK pathway.

Key words: Psoriasis, EPHA2, Keratinocyte, Cell proliferation, Cell differentiation, ERK pathway

引用本文

蒋星宇, 余增洋, 马蕊, 时熔灿, 黄大炜, 王媛媛, 蔡江鲁伊, 史玉玲, . 银屑病中EPHA2通过ERK通路调控角质形成细胞增殖分化的机制研究[J]. 中华皮肤科杂志, 2025,58(11):1042-1052. doi:10.35541/cjd.20240655

Jiang Xingyu, Yu Zengyang, Ma Rui, Shi Rongcan, Huang Dawei, Wang Yuanyuan, Cai Jiangluyi, Shi Yuling, . Investigations into the mechanisms underlying the regulatory effect of EPHA2 on keratinocyte proliferation and differentiation via ERK pathway in psoriasis[J]. Chinese Journal of Dermatology, 2025, 58(11): 1042-1052.doi:10.35541/cjd.20240655