中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (8): 569-573.

• 论著 • 上一篇    下一篇

荷人皮肤鳞状细胞癌裸鼠移植瘤模型中干扰血管内皮生长因子的实验研究

曹一鑫1,冯新2,王建力1,陈莉3   

  1. 1. 南通大学附属医院皮肤科
    2. 江苏省海门市皮肤病防治研究所
    3. 南通大学医学院
  • 收稿日期:2011-08-18 修回日期:2012-03-08 出版日期:2012-08-15 发布日期:2012-08-01
  • 通讯作者: 陈莉 E-mail:guilan@ntu.edu.cn
  • 基金资助:

    南通市社会发展基金

Suppression of vascular endothelial growth factor expression by RNA interference in human skin squamous cell carcinoma xenografts in nude mouse models

1, 1, li chen lichen   

  • Received:2011-08-18 Revised:2012-03-08 Online:2012-08-15 Published:2012-08-01
  • Contact: li chen lichen E-mail:guilan@ntu.edu.cn

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摘要：

目的 在沉默血管内皮生长因子（VEGF）的荷人裸鼠皮肤鳞状细胞癌（鳞癌）移植瘤中观察肿瘤生长，探讨靶向VEGF小发夹核酸（shRNA）的作用。方法 生物合成靶向人VEGF基因的shRNA干扰真核表达质粒（psilencer-VEGF1-shRNA、VEGF-s1；psilencer-VEGF2-shRNA、VEGF-s2）,同时合成含随机靶序列的阴性对照表达质粒（psilencer-Target-off-shrank，T-off）。将构建的质粒分别转染于筛选的人皮肤鳞癌细胞株（A431），获得稳转细胞株。实时荧光定量逆转录-聚合酶链反应、双抗体夹心酶联免疫吸附法分别检测稳转细胞株中VEGF mRNA和VEGF蛋白的表达。用稳转细胞株制备荷人皮肤鳞癌裸鼠移植瘤模型，观察裸鼠体内肿瘤生长，6周后（20 d）处死裸鼠进行肿瘤病理学研究，免疫组化染色检测瘤组织VEGF、增殖细胞核抗原（PCNA）和CD34蛋白的表达。应用stata 7.0统计学软件进行统计学处理。组间比较采用t检验。结果 转染VEGF-s1和VEGF-s2的A431细胞中VEGF mRNA表达分别为27.85 ± 3.95和24.69 ± 2.83，表达量显著低于未转染组（54.06 ± 6.38，t值分别为6.05和7.29，P值均 ＜ 0.01）；VEGF蛋白表达分别为32.67 ± 2.52和29.27 ± 1.10，亦显著低于未转染组（52.85 ± 2.23，t值分别为8.04和11.53，P值均 ＜ 0.01）。用转染VEGF-s1和VEGF-s2的A431细胞制备荷人皮肤鳞癌裸鼠移植瘤模型，裸鼠肿瘤体积分别为（192.50 ± 10.90） mm3和（203.67 ± 3.21） mm3，明显小于未转染组（272.00 ± 21.07 mm3，t值分别为5.80和5.55，P值均 ＜ 0.01）；裸鼠肿瘤重量分别为（0.05 ± 0.03） g和（0.13 ± 0.04） g，与未转染组（0.25 ± 0.02 g）比较明显减轻（t值分别为9.60和4.64，P值均 ＜ 0.01）；裸鼠肿瘤细胞中VEGF蛋白表达率分别为52.00% ± 2.00%和56.67% ± 3.06%，PCNA阳性率分别为37.01% ± 2.41%和33.94％ ± 3.25%，CD34阳性血管数分别为2.05 ± 0.07和1.72 ± 0.10，与未转染组（70.00% ± 2.00%、72.11% ± 3.02%和4.01 ± 1.27）比较，均显著降低（P值均 ＜ 0.01）。各项指标中，转染VEGF-s1和VEGF-s2组间、未转染组和T-off组间差异均无统计学意义（P ＞ 0.05）。结论 靶向VEGF基因的shRNA能有效抑制A431细胞和荷人裸鼠皮肤鳞癌移植瘤中VEGF的表达，导致肿瘤生长受抑，肿瘤恶性表型减弱。

关键词: VEGF

Abstract:

Objective To observe the effect of short hairpin RNA （shRNA）-mediated vascular endothelial growth factor （VEGF） gene silencing on the growth of human skin squamous cell carcinoma（SCC） xenografts in nude mice. Methods Two eukaryotic expression plasmids targeting VEGF gene, including psilencer-VEGF1-shRNA （VEGF-s1） and psilencer-VEGF2-shRNA （VEGF-s2）, as well as one negative control plasmid containing random target sequence （psilencer-Target-off-shRNA, T-off）, were chemically synthesized, and transfected into a human skin SCC cell line A431 to develop stably transfected cell lines. Real time quantitative PCR （RT-qPCR） and double-antibody sandwich enzyme-linked immunosorbent assay （ELISA） were carried out to measure the expression of VEGF mRNA and protein respectively in A431 cells. Twelve nude mice were divided into 4 goups to be subcutaneously inoculated in the axillary region with untransfected A431 cells as well as A431 cells transfected with VEGF-s1, VEGF-s2 and T-off, respectively. The tumor growth was observed in nude mice every 5 days. Twenty days after the inoculation, the mice were sacrificed, and transplanted tumors were obtained from the mice and subjected to an immunohistochemical study for the measurement of VEGF，proliferating cell nuclear antigen （PCNA） and CD34 expression. Data were statistically analyzed by using the Stata 7.0 software, and t test was conducted to compare the differences between groups. Results The mRNA and protein expression levels of VEGF were significantly lower in A431 cells transfected with VEGF-s1 and VEGF-s2 than in untransfected A431 cells （27.85 ± 3.95 and 24.69 ± 2.83 vs. 54.06 ± 6.38, t = 6.05, 7.29, both P ＜ 0.01; 32.67 ± 2.52 and 29.27 ± 1.10 vs. 52.85 ± 2.23, t = 8.04 and 11.53, both P ＜ 0.01）. Twenty days after the inoculation, the volume and weight of xenografted tumors in mice inoculated with VEGF-s1- and VEGF-s2-transfected A431 cells were significantly lower than those in mice with untransfected A431 cells （（192.50 ± 10.90） mm3 and （203.67 ± 3.21） mm3 vs. （272.00 ± 21.07） mm3, t = 5.80 and 5.55, both P ＜ 0.01; （0.05 ± 0.03） g and （0.13 ± 0.04） g vs. （0.25 ± 0.02） g, t = 9.60 and 4.64, both P ＜ 0.01）. Decreased expression rate of VEGF, PCNA and number of CD34-positive vessels were observed in the xenografted tumor tissue from mice inoculated with VEGF-s1- and VEGF-s2-transfected A431 cells compared with that from mice with untransfected A431 cells （52.00% ± 2.00% and 56.67% ± 3.06% vs. 70.00% ± 2.00%，both P < 0.01; 37.01% ± 2.41% and 33.94% ± 3.25% vs. 72.11% ± 3.02%, both P < 0.01; 2.05 ± 0.07 and 1.72 ± 0.10 vs. 4.01 ± 1.27, both P < 0.01）. No significant differences were observed in the above parameters between cells transfected with VEGF-s1- and VEGF-s2-transfected A431 cells, between untransfected and T-off-transfected A431 cells, between tumor xenografts derived from VEGF-s1- and VEGF-s2-transfected A431 cells, or between tumor xenografts derived from untransfected and T-off-transfected A431 cells （all P > 0.05）. Conclusions The shRNA targeting VEGF gene can significantly inhibit the expression of VEGF in A431 cells and A431-derived tumor xenografts in nude mice, in turn suppress the growth and attenuate the malignant phenotype of tumor.

Key words: VEGF