中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (7): 501-504.

• 论著 • 上一篇    下一篇

内皮素3对恶性黑素瘤A375细胞上皮基质转化的影响

李艳秋1,朱里2,曾山鹰3,王翠彦4,孙兰3,4,林云2,陈宏翔5,黄长征4,陈思远6   

  1. 1. 湖北省武汉市协和医院皮肤科
    2. 武汉华中科技大学同济医学院附属协和医院皮肤科
    3.
    4. 华中科技大学同济医学院附属协和医院皮肤科
    5. 华中科技大学同济医学院附属协和医院
    6. 武汉市华中科技大学同济医学院附属协和医院皮肤科
  • 收稿日期:2011-07-08 修回日期:2012-02-03 出版日期:2012-07-15 发布日期:2012-07-02
  • 通讯作者: 黄长征 E-mail:hcz0501@126.com
  • 基金资助:

    内皮素3在黑素瘤发病机制中的研究;CTHRC1在恶性黑素瘤侵袭及转移机制中的研究;TGF-B对鼠B16细胞上皮基质转化的影响

Effects of endothelin-3 on epithelial-to-mesenchymal transition in a malignant melanoma cell line A375

  • Received:2011-07-08 Revised:2012-02-03 Online:2012-07-15 Published:2012-07-02
  • Contact: HUANG Chang-Zheng E-mail:hcz0501@126.com

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摘要：

目的 研究内皮素-3（ET-3）对人恶性黑素瘤（MM）A375细胞上皮基质转化（EMT）的影响。方法 体外培养A375细胞，分别设立3组：空白对照组、100 nmol/L ET-3组、100 nmol/L ET-3和100 μmol/L BQ788（内皮素受体B阻断剂）组。采用Transwell小室检测细胞转移，细胞爬片技术检测细胞形态变化，实时PCR和Western印迹检测上皮基质转化相关分子上皮细胞钙黏蛋白、波形蛋白及转录因子（Twist、 Slug）表达情况，使用方差分析及Scheffe法对结果进行分析。结果 各干预条件中，与空白对照组比较，ET-3可以促进A375细胞的转移，BQ788可阻断该效应（3组穿膜细胞数分别为4.200 ± 0.837、9.400 ± 0.548、3.400 ± 0.894，F = 88.44，P < 0.01）；ET-3 可以促进A375细胞由上皮型向成纤维细胞样形态转变，促进A375上皮细胞钙黏蛋白表达下调（3组分别为0.330 ± 0.002、0.280 ± 0.007、0.420 ± 0.008，F = 329.98，P < 0.01），波形蛋白表达上调（0.830 ± 0.014、1.160 ± 0.003、0.750 ± 0.030，F = 262.94，P < 0.01），而BQ788可阻断这种效应。ET-3可以促进上皮基质转化相关转录因子Slug mRNA（F = 376.94，P < 0.01）及Twist mRNA（F = 215.62，P < 0.01）及其蛋白水平（FSlug = 288.87，P < 0.01；FTwist = 156.96，P < 0.05）上的表达上调。结论 ET-3/ETRB通过上调波形蛋白，下调上皮细胞钙黏蛋白的表达，并上调转录因子（Twist、 Slug）的表达，促进黑素瘤A375细胞上皮基质转化。

关键词: 上皮基质转化

Abstract:

Objective To explore the role of endothelin-3 （ET-3） on epithelial-to-mesenchymal transition in a malignant melanoma cell line A375. Methods A375 cells were cultured in vitro and classified into 3 groups to be treated with ET-3 at 100 nmol/L （ET-3 group）, co-cultured with ET-3 at 100 nmol/L and endothelin receptor B （ETRB） antagonist BQ788 at 100 μmol/L （ETRB antagonist group）, or to remain untreated （blank control group）. After additional 24-hour culture, Transwell chamber assay was used to detect the invasive capability of A375 cells, real time-PCR to measure the mRNA expressions of Twist and Slug, and Western blot to determine the protein expression of E-cadherin, vimentin, Twist and Slug. The changes in the morphology of A375 cells were observed. Data were assessed by analysis of variance and Scheffe's method. Results In the Transwell assay, the number of A431 cells permeating through the basement membrane was 4.200 ± 0.837, 9.400 ± 0.548 and 3.400 ± 0.894 respectively in the blank control group, ET-3 group and ETRB antagonist group （F = 88.44, P < 0.01）, suggesting that ET-3 could promote the metastasis of A375 cells, while BQ788 could block the promotive effect of ET-3. The epithelial-to-mesenchymal transition was obvious in cells treated with ET-3 alone, but was inapparent in cells treated with ET-3 and BQ788. The ET-3 at 100 nmol/L significantly decreased the protein expression of E-cadherin from 0.33 ± 0.002 （blank control group） to 0.28 ± 0.007，but increased that of vimentin from 0.83 ± 0.014 （blank control group） to 1.16 ± 0.003, while BQ788 upregulated the E-cadherin expression to 0.42 ± 0.008 and downregulated the vimentin expression to 0.75 ± 0.030, and significant differences were observed in the E-cadherin expression and vimentin expression among the ET-3 group, ETRB antagonist group and blank control group （F = 329.98，262.94，respectively, both P < 0.01）. A significant increase was observed in the mRNA and protein expression of Slug （F = 376.94, 288.87, both P < 0.01） and Twist （F = 215.62, 156.96, P < 0.01 and 0.05） in A375 cells after treatment with ET-3. Conclusion ET-3/ETRB axis may promote the epithelial-to-mesenchymal transition in A375 cells likely by regulating the expression of E-cadherin, vimentin and two important transcription factors Twist and Slug.

Key words: Epithelial-to-Mesenchymal Transition

中图分类号: 

  • R739.5