腺病毒介导RNA干扰抑制人结肠癌VEGF受体表达及肿瘤生长

Adenovirus vector-mediated RNA interference inhibits expression of vascular endothelial growth factor receptor in colon carcinoma cells and tumor growth

目的:观察腺病毒介导的RNA干扰抑制人结肠癌中血管内皮细胞生长因子受体(VEGFR)表达及肿瘤生长的作用。方法:将RNA干扰pAd-Easy/VEGFR腺病毒载体用Lipofectamine 2000转染293细胞,制备携带人VEGFR的腺病毒,转染CW2细胞,通过荧光显微镜和流式细胞仪观察转染效率,通过RT-PCR和蛋白质印迹法检测VEGFR mRNA的表达;以MTT比色法测定活细胞数并绘制细胞生长曲线。同时,制备裸鼠CW2细胞移植瘤模型,注射腺病毒后,每天观察肿瘤生长情况。应用免疫组化法检测微血管密度(MVD)。结果:pAd-Easy/VEGFR重组腺病毒成功构建,转染效率为99.7%。RT-PCR、Western印迹实验结果显示转染pAd-Easy/VEGFR后,VEGFR的表达明显被抑制。细胞生长曲线表明,重组载体转染后细胞生长明显减缓。体内实验表明,pAd-Easy/VEGFR治疗后肿瘤生长慢并伴有较低的MVD。结论:pAd-Easy/VEGFR介导的VEGFR shRNA能有效抑制CW2细胞中VEGFR的表达,并能抑制肿瘤生长。

Objective：To study the inhibitory effects of an adenovirus （Ad）-based short hairpin RNA （shRNA） expression system on expression of vascular endothelial growth factor receptor （VEGFR） and on growth of colon carcinoma cells in vitro and in vivo. Methods： RNA interference pAd-Easy/VEGFR vector was used to transfect 293 cells via Lipofectamine 2000. The adenoviral vector carrying VEGFR was used to transfect CW2 cells and the transfection efficiency was determined by fluorescent microscope and flow cytometry. The expression of VEGFR was examined by RT-PCR and Western blotting. The cell growth was observed with MTT method and the growth curve was plotted. Nude mouse was transplanted with CW2 cells to establish colon cancer tumor model and the growth of tumor was observed daily. Micro vessel density （MVD） was detected by immunohistochemistry. Results： The recombinant pAd-Easy carrying shRNA against VEGFR was verified by sequencing. Flow cytometry showed that the transfection efficiency of CW2 cells was 99. 7M. RT-PCR and Western blotting showed that the expression of VEGFR in pAd-Easy/VEGFR group was obviously decreased. The growth curve showed that the cell growth in the pAd-Easy/ VEGFR group was obviously slowed down. We also found that the tumor growth in the nude mouse model was obviously inhibited and the MVD was also decreased. Conclusion： pAd-Easy/VEGFR-mediated VEGFR shRNA can effectively inhibit the expression of VEGFR in CW2 cells and suppress tumor growth in vivo.