中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (12): 877-882.

• 论著 • 上一篇    下一篇

过表达表皮生长因子受体对人类黑素瘤脑转移瘤细胞系H1生长和转移的影响

康晶1,Hrvoje Miletic2,郭淑兰3   

  1. 1. 山东大学
    2. University of Bergen
    3. 山东大学齐鲁医院皮肤科
  • 收稿日期:2014-05-02 修回日期:2014-09-18 出版日期:2014-12-15 发布日期:2019-06-14
  • 通讯作者: 郭淑兰 E-mail:guoshulan@medmail.com.cn

Effect of overexpression of epidermal growth factor receptor on the growth and migration of a brain-metastatic H1 human melanoma cell line

  • Received:2014-05-02 Revised:2014-09-18 Online:2014-12-15 Published:2019-06-14

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摘要： 目的 探索表皮生长因子受体（EGFR）在黑素瘤生长和转移过程中的作用。 方法 选用本实验室已建立的人黑素瘤脑转移瘤细胞系（H1），通过慢病毒载体稳定转染野生型EGFR（H1EGFRwt），使其呈EGFR过表达状态，对照组转染绿色荧光蛋白（H1GFP）后分选纯化。采用细胞划痕实验对实验组H1EGFRwt细胞和对照组H1GFP细胞的迁移能力进行分析，结果以划痕面积愈合率表示。采用集落形成实验观察EGFR对两组细胞独立生存能力和集落形成能力的影响，通过刃天青还原试验评估集落形成结果，结果以两组细胞活细胞数及其代谢活性评分表示。Western印迹分析EGFR激活的信号通路。采用SPSS 20.0软件进行重复测量的方差分析比较两组细胞划痕面积愈合率的差异，采用配对样本t检验比较两组细胞不同预处理的代谢评分差异。 结果 流式细胞仪检测显示，H1细胞EGFR和GFP转染成功，分选后得到了纯化H1EGFRwt和H1GFP细胞。细胞划痕实验显示，实验组H1EGFRwt在给予表皮生长因子（EGF）刺激的不同时间点（6、18、24、48 h），划痕面积愈合率分别为0.145 ± 0.066、0.479 ± 0.096、0.571 ± 0.198、0.672 ± 0.199，对照组H1GFP在相同条件下分别为0.051 ± 0.036、0.254 ± 0.038、0.303 ± 0.077和0.498 ± 0.111， H1EGFRwt细胞的愈合程度均高于H1GFP细胞，两组比较，F = 68.49，df = 5，P < 0.05。集落形成实验证实，H1EGFRwt细胞的代谢评分（92 225.2 ± 6 632.1）高于H1GFP细胞（62 935.7 ± 8 159.2），两组比较，t = 2.26，df = 9，P < 0.01。Western印迹结果显示，H1EGFRwt细胞可在EGF的刺激下激活下游信号磷酸化磷脂酰肌醇特异性磷脂酶C-γ（pPLCγ）、磷酸化信号转导和转录活化因子5（pSTAT 5）、磷酸化信号转导和转录活化因子3（pSTAT3），而对照组未能激活。此外，H1EGFRwt细胞可在EGF刺激下使磷酸化丝/苏氨酸蛋白激酶（pAKT）和磷酸化有丝裂原活化蛋白激酶（pMAPK）表达明显高于对照组。 结论 EGFR在黑素瘤H1细胞的转移机制中发挥重要作用，并有可能成为黑素瘤转移治疗中的靶向目标。

关键词: 黑色素瘤, 受体，表皮生长因子, 表皮生长因子

Abstract: Kang Jing*, Hrvoje Miletic, Guo Shulan. *Shandong University School of Medicine, Jinan 250012, China Corresponding author: Guo Shulan, Email: guoshulan@medmail.com.cn 【Abstract】 Objective To investigate the role of epidermal growth factor receptor （EGFR） in the growth and migration of melanoma. Methods A brain-metastatic human melanoma cell line H1, which had been developed in our laboratory, was used in this study. Some H1 melanoma cells were stably transfected with wild-type EGFR via lentiviral vectors to overexpress EGFR （H1EGFRwt）, and some H1 cells transfected with green fluorescent protein （GFP） served as the control group （H1GFP）. After transfection, the cells were sorted and purified. Then, scratch wound healing assay was performed to estimate the migratory activity of H1 cells; colony-forming assay was conducted to investigate the effects of EGFR on the survival ability and colony-forming ability of H1 cells, and resazurin reduction test was used to evaluate colony formation results; Western blot was carried out to measure the expressions of signaling pathways activated by EGFR. Statistical analysis was done by repeated measures analysis of variance （ANOVA） for the comparison of wound-area healing rate, and by paired t test for the comparison of metabolic activity, between the two groups of H1 cells by using SPSS software version 20.0. Results Flow cytometry showed that H1 cells were successfully transfected with EGFR and GFP respectively, and purified H1EGFRwt and H1GFP cells were obtained after cell sorting. The wound-area healing rate at 6, 18, 24 and 48 hours after epidermal growth factor （EGF） stimulation was 0.145 ± 0.066, 0.479 ± 0.096, 0.571 ± 0.198 and 0.672 ± 0.199 respectively for H1EGFRwt cells, 0.051 ± 0.036, 0.254 ± 0.038, 0.303 ± 0.077 and 0.498 ± 0.111 respectively for H1GFP cells. The degree of wound healing in H1EGFRwt cells was significantly higher than that in H1GFP cells （F = 68.49, df = 5, P < 0.05）. Colony-forming assay revealed that the average metabolic activity score of H1EGFRwt cells was significantly higher than that of H1GFP cells （92 225.2 ± 6 632.1 vs. 62 935.7 ± 8 159.2, t = 2.26, df = 9, P < 0.01）. Western blot showed that EGF induced the phosphorylation of downstream signaling proteins such as phosphatidylinositol-specific phospholipase C-γ （PLCγ）, signal transducer and activator of transcription 5 （STAT5） and 3 （STAT3） in H1EGFRwt cells, but not in H1GFP cells. In addition, the expressions of phosphorylated serine/threonine protein kinase （pAKT） and mitogen-activated protein kinase （pMAPK） were significantly higher in H1EGFRwt cells than in H1GFP cells after EGF stimulation. Conclusions EGFR may play an important role in the metastasis of H1 melanoma cells, and might serve as a target in the treatment of metastatic melanoma.

Key words: Melanoma, Receptor, epidermal growth factor, Epidermal growth factor