中华皮肤科杂志 ›› 2009, Vol. 42 ›› Issue (10): 711-714.

• 论著 • 上一篇    下一篇

腺病毒载体介导的内皮抑素基因治疗小鼠黑素瘤的实验研究

曹瑞华1,廖万清2,温海3,刘翠杰4   

  1. 1. 济南军区总医院皮肤科济南市师范路25号 250031
    2. 上海第二医科大学长征医院皮肤科
    3. 上海第二军医大学长征医院皮肤科
    4. 济南军区总医院皮肤科
  • 收稿日期:2008-11-04 修回日期:2008-12-04 发布日期:2009-10-09
  • 通讯作者: 曹瑞华 E-mail:ruihuacao@sohu.com
  • 基金资助:

    无

Adenovirus-mediated human endostatin gene delivery in the treatment of mouse melanoma

  • Received:2008-11-04 Revised:2008-12-04 Published:2009-10-09

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摘要：

目的　探讨腺病毒载体介导的内皮抑素基因（Ad-mES）在体外和体内的生物学活性。方法　不同感染复度（MOI）的腺病毒体外感染靶细胞；RT-PCR法检测目的基因的表达；MTT法检测Ad-mES对靶细胞生物活性的影响。观察各组小鼠黑素瘤的生长、转移和生存率；免疫组化法鉴定肿瘤组织内内皮抑素蛋白的表达。电子透射电镜观察肿瘤组织内皮细胞、肿瘤细胞的凋亡情况。结果　腺病毒体外能够有效感染靶细胞，MOI为10，20，50，100，200，500时，B16F10细胞和ECV304细胞的腺病毒感染率分别为15.6%、35%、73%、88%、95.2%、97%和19%、35%、80%、90%、97%、98.5%。靶细胞明确表达内皮抑素基因；Ad-mES对B16F10细胞的增殖没有影响；而Ad-mES能抑制ECV304细胞的增殖，且随MOI增大，抑制内皮细胞增殖效果越强。瘤细胞接种后第8天，各组成瘤率100%。开始出现小鼠死亡的最早日：PBS组第16天、Ad-GFP组第18天、Ad-mES单剂、重复治疗组均在第20天。结论 Ad-mES体外和体内均影响靶细胞的生物学活性；Ad-mES治疗组小鼠平均生存时间延长（P < 0.05），肿瘤体积增长减慢（P < 0.05）。

关键词: 内皮抑素, 腺病毒载体, 基因治疗, 血管生成, 黑色素瘤

Abstract:

Objective To observe the bioactivity of adenovirus-mediated human endostatin gene in vivo and in vitro. Methods B16F10 melanoma cells and human endothelial cells （ECV 304） were both transfected with recombinant adenovirus containing green fluorescent protein （Ad-GFP） or human endostatin gene （Ad-mES） at various multiplicity of infection （MOI）. Then, the expression of endostatin gene was detected by RT-PCR, and the growth of cells by MTT assay. B16F10 cells were inoculated into the back of mice to establish melanoma models, which were classified into treated groups intratumorally injected with Ad-mES once （single Ad-mES group） or repeatedly （repetitive Ad-mES group） with an interval of 7 days, and control groups intratumorally injected with Ad-GFP （Ad-GFP group） or phosphate buffered solution （PBS group）. Subsequently, the growth of tumors was observed at an interval of 4 days; tumor tissue samples were obtained from killed mice and subjected to the detection of endostatin expression with immunohistochemistry on day 7 after the first intratumoral injection; transmission electron microscopy was used to observe the apoptosis of tumor cells and endothelial cells on day 14. Results Adenovirus could efficiently infect targeted cells. When the MOI of Ad-GFP was 10, 20, 50, 100, 200 and 500, B16F10 cells were infected at a rate of 15.6%, 35%, 73%, 88%, 95.2% and 97%, respectively, and ECV304 cells at a rate of 19%, 35%, 80%, 90%, 97% and 98.5%, respectively. The endostatin gene was proved to be expressed in targeted cells. Ad-mES had no obvious effect on B16F10 cells, but inhibited the growth of ECV 304 cells, and the inhibitive effect was enhanced with the concentration of Ad-mES. The rate of tumor formation was 100% on day 8 after injection of B16F10 cells, and the earliest death of mice was observed on day 16 in PBS group, day 18 in Ad-GFP group, day 20 in single Ad-mES group and repetive Ad-mES group. Conclusions The recombinant Ad-mES could affect the bioactivity of targeted cells in vivo and in vitro. Intratumoral injection of Ad-mES prolonged the survival of mice bearing melanoma and deccelarated the growth of melanoma.