摘要
目的:用RNA干扰技术阻断人舌鳞癌系Tca8113细胞中VEGF基因的表达,观察细胞增殖及凋亡特征。方法:检测人舌鳞癌系Tca8113细胞中血管内皮生长因子VEGF表达水平;采用真核转录质粒pRNA-U6.1/Neo构建针对VEGF基因的重组转染质粒。将4种不同序列的VEGF-shRNAV1、VEGF-shRNAV2、VEGF-shRNAV3、VEGF-shRNAV4质粒以及含与VEGF无关序列的VEGF-shRNAVc和空白质粒VEGF-shRNAU6分别转染Tca8113细胞,48h后检测其VEGF表达水平,筛选稳定转染RNAi-VEGF的细胞;CCK8检测其增殖能力;流式细胞仪检测其细胞周期;Anexin-V-PI检测其凋亡特征。结果:RT-PCR和Western blot检测:Tca8113细胞阳性表达VEGF;用Lipofectamine 2000成功将不同VEGF片段的质粒转染入Tca8113细胞;RT-PCR发现,VEGF-shR-NAV2组细胞VEGF表达被显著抑制,用400ng/mLG418压力筛选获得VEGF稳定被抑制的细胞系;CCK8检测各组Tca8113细胞增殖见VEGF-shRNAV2组增殖能力小于VEGF-shRNAVc组和VEGF-shRNAU6组;转染VEGF-shRNAV2的Tca8113细胞G0-G1期细胞较VEGF-shRNAVc组和VEGF-shRNAU6组高,且细胞凋亡多。结论:用RNA靶向干扰能明显降低人舌鳞癌Tca8113细胞内VEGF的表达并抑制肿瘤细胞增殖及促进凋亡。
Objective：To construct the recombined plasmid expressing VEGF short hairpin RNA （shRNA） by pRNA-U6.1/Neo plasmid vector and observe the proliferation and the apoptosis of Tca8113 cells after RNAi-mediated VEGF gene silencing. Methods：The expression of vascular endothelial growth factor （VEGF） was detected by RT-PCR and Western blot. There were six recombination eukaryotic expression vectors. They were 4 kinds vectors including differenr VEGF sequences and 2 kinds of blank vectors including irrespective sequence and of no sequence. After they were transfected into the Tca8113 cells by Lipofectamine 2000,the interference effect was detected by RT-PCR after 48h. The tranfected Tca8113 cells expressing low level VEGF namely VEGF-shRNAV2 were selected by G418. Cell count kit 8 was used to detect the proliferation of VEGF-shRNAV2 and the cell cycle was detected by flow cytometry. The apoptosis was detected by AnexinV-PI. Results：The expression of VEGF on Tca8113 cells was detected by RT-PCR and Western blot. The efficiency of transfection was high by observe the expression of GFP. The VEGF expression of VEGF-shRNAV2 was lower than others,and it was selected as the experiment cell line. The proliferative ability of VEGF-shRNAV2 was lower than those of VEGF-shRNAVc and VEGF-shRNAU6. The G0-G1 proportion and the apoptosis of those of VEGF-shRNAV2 was higher than VEGF-shRNAVc and VEGF-shRNAU6. Conclusion：Targeting the expression of VEGF on human tongue squamous cell carcinoma cells Tca8113 using RNA interference can significantly reduce and inhibit the cell proliferation and promote the cell apoptosis.
出处
《口腔医学研究》
CAS
CSCD
北大核心
2009年第2期124-128,共5页
Journal of Oral Science Research
基金
国家自然科学基金资助(编号:30772441)
关键词
口腔鳞状细胞癌
血管内皮生长因子
RNA干扰
质粒
Oral squamous cell carcinoma Vascular endothelial growth factor RNA interference Plasmid
