中华皮肤科杂志 ›› 2005, Vol. 38 ›› Issue (5): 306-308.

• 论著 • 上一篇    下一篇

血管内皮生长因子-siRNA对恶性黑素瘤细胞生长的抑制作用

涂亚庭1, 陶娟1, 黄长征1, 张杏平2, 张丽霞1, 沈关心3   

  1. 1. 华中科技大学同济医学院附属协和医院皮肤科, 武汉430022;
    2. 华中科技大学同济医学院附属同济医院皮肤科;
    3. 华中科技大学同济医学院免疫学教研室
  • 收稿日期:2004-05-15 出版日期:2005-05-15 发布日期:2005-05-15
  • 通讯作者: 涂亚庭,E-mail:tjhappy@126.com E-mail:tjhappy@126.com

Inhibition of Malignant Melanoma Cell Growth Through Blocking Expression of VEGF by RNA Interference

TU Ya-ting1, TAO Juan1, HUANG Chang-zheng1, ZHANG Xing-ping2, ZHANG Li-xia1, SHEN Guan-xin3   

  1. Department of Dermatology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
  • Received:2004-05-15 Online:2005-05-15 Published:2005-05-15

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摘要： 目的 探讨血管内皮生长因子siRNA对恶性黑素瘤细胞株A375增殖和凋亡的影响。方法 构建针对血管内皮生长因子的siRNA真核表达载体pU-VEGF-siRNA将其表达载体经电穿孔转染A375细胞,采用逆转录聚合酶链反应(RT-PCR)、免疫酶联吸附实验(ELISA)方法 检测转染后的A375细胞VEGF的mRNA和蛋白表达,应用细胞计数比较转染组和对照组A375细胞增殖能力,将含转染组和对照组A375细胞的上清液分别培养人静脉内皮细胞(ECV-304),观察其生长情况,并用流式细胞仪观察转染pU-VEGF-siRNA载体对A375细胞凋亡的影响。结果 转染pU-VEGF-siRNA载体后,A375细胞的VEGFmRNA和蛋白表达均较对照组明显下降,并且其生长缓慢,其细胞周期出现亚二倍凋亡峰。用转染pU-VEGF-siRNA的A375细胞上清液培养的ECV-304细胞增殖力较对照组下降。结论 通过RNA干扰技术阻断VEGF的表达,可抑制A375和ECV-304细胞增殖,诱导A375细胞凋亡。

关键词: 黑色素瘤, 内皮生长因子, siRNA, 细胞凋亡

Abstract: Objectives To construct a small interfering RNA (siRNA) targeting vascular endothelial growth factor (VEGF) in eukaryotic expression vector, and to assess effects of this vector on proliferation and apoptosis of malignant melanoma cell line A375. Methods pU-VEGF-siRNA plasmid was transfected into A375 cells by electroporation. VEGF mRNA and protein were detected by reverse transcription polymerse chain reaction (RT-PCR) and ELISA, respectively, in A375 cells after gene transfer. Proliferation of A375 cells was assessed by cell counting. Human umbilical vein endothelial cells (ECV-304) were cultured in medium containing supernatants of treated and untreated A375 cells, and their viability was tested by cell counting. Apoptosis of A375 cells was observed by flow cytometry (FCM). Results VEGF mRNA and protein were significantly decreased in the experimental group, compared to those in the controls (P<0.05). Proliferation of treated A375 and ECV-304 cells was inhibited. Subdiploidy peaks were also found in the treated A375 cells. Conclusions Delivery of siRNA targeting VEGF seems efficient in down-regulating VEGF expression, in inhibiting A375 and ECV-304 cell proliferation and in inducing A375 cell apoptosis, suggesting that siRNA-based strategy targeting VEGF strategy may lay a foundation for clinical management of malignant melanoma.

Key words: Melanoma, Endothelial growth factors, siRNA, Apoptosis