中华皮肤科杂志

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pU-VEGF-siRNA对裸鼠恶性黑素瘤生长影响的研究

陶娟1, 涂亚庭1, 林云1, 沈关心2   

  1. 1. 华中科技大学同济医学院附属协和医院皮肤科, 430022;
    2. 华中科技大学同济医学院免疫学系
  • 收稿日期:2005-06-13 出版日期:2006-03-15 发布日期:2006-03-15
  • 通讯作者: 涂亚庭, email:tjhappy@126.com E-mail:tjhappy@126.com
  • 基金资助:
    国家自然科学基金资助项目(30500437)

In vivo studies on the role of pU-VEGF-siRNA in the growth of malignant melanoma

TAO Juan1, TU Ya-ting1, LIN Yun1, SHEN Guan-xin2   

  1. Department of Dermatology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
  • Received:2005-06-13 Online:2006-03-15 Published:2006-03-15

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摘要： 目的 研究pU-VEGF-siRNA对恶性黑素瘤成瘤和凋亡的影响及其机制.方法 构建针对血管内皮生长因子(VEGF)的发卡样siRNA真核表达载体pU-VEGF-siRNA,通过电穿孔法将构建重组体导入人恶性黑素瘤细胞系A375,并建立pU-VEGF-siRNA转染细胞荷瘤裸鼠模型,应用免疫组织化学方法检测荷瘤裸鼠肿瘤组织VEGF和血管内皮细胞特异性Ⅷ因子相关抗原(FⅧRAg)的表达,依据FⅧRAg的表达评价肿瘤微血管密度,末端脱氧核苷酸转移酶标记法(TUNEL)定量检测荷瘤裸鼠模型的肿瘤组织凋亡.结果 体内实验表明,实验组成瘤率明显低于对照组,且其肿瘤生长速度也明显减慢(P<0.01).实验组VEGF表达和肿瘤微血管密度明显低于对照组(P<0.01).实验组可见大量凋亡细胞,对照组仅见少许凋亡细胞,实验组凋亡指数与对照组相比差异有统计学意义(P<0.01).结论 通过RNA干扰技术阻断VEGF的表达,在裸鼠体内可显著抑制恶性黑素瘤生长.

关键词: 黑色素瘤,实验性, pU-VEGF-siRNA, 细胞凋亡

Abstract: Objectives To study the effects of pU- vascular endothelial growth factor (VEGF) short interfering RNA (siRNA) on the formation and apoptosis of malignant melanoma in models of nude mice and its mechanism. Methods The siRNA eukaryotic expression vector for VEGF was constructed, then transfected into A375 (a human malignant melanoma cell line) by electroporation. The nude mice models of malignant melanoma were constructed. The protein expression of VEGF and factor Ⅷ related antigen (FⅧRAg) specific for vascular endothelial cells was detected by immunohistochemical technique and morphological quantitative analysis. Microvessel density (MVD) was counted based on the endothelial cells positively stained with anti-FⅧRAg antibody. The apoptosis of the neoplasms in nude mice was quantitatively determined by TdT mediated dUTP nick end labeling (TUNEL). Results Both the number and the growth speed of the neoplasm formation were lower in the experimental group than those in the controls (both P<0.01). VEGF expression and MVD significantly decreased in experimental group than those in the controls (P<0.01). Numerous apoptotic cells were found in the experimental groups,but few in the controls. The apoptotic indices were significantly higher in the experimental group than those in the controls (P<0.01). Conclusions The delivery of siRNA directed against VEGF is shown to inhibit the growth of malignant melanoma in vivo, suggesting that siRNA-based, VEGF targeting strategy might benefit the research of gene therapy for malignant melanoma.

Key words: Melanoma,experimental, pU-VEGF-siRNA, Apoptosis