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。

abstractsObjective: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.