摘要
目的:构建靶向肝癌HepG2细胞VEGF基因的siRNA表达载体并在体外检测其对VEGF基因表达的抑制作用.方法:设计合成靶向HepG2细胞VEGF基因的siRNAcDNA序列并与pSUPER载体连接,构建VEGF siRNA表达载体,经酶切鉴定和测序确认后,脂质体2000介导VEGF siRNA转染HepG2细胞.RT-PCR及Western blot检测VEGF siRNA表达载体转染的HepG2细胞中VEGF基因表达.结果:通过双酶切鉴定和测序分析,成功构建了靶向HepG2细胞VEGF基因的siRNA表达载体.RT-PCR和Western blot法检测转染VEGF siRNA表达载体的HepG2细胞VEGF mRNA及VEGF165表达下调,其抑制率分别为65%和74%.实验组中的HepG2中VEGF mRNA表达较阴性对照组、空载体组表达量显著下降(0.304±0.062vs0.896±0.061,0.884±0.074,P<0.05).结论:成功构建了靶向肝癌HepG2细胞VEGF基因的siRNA表达载体,且该载体在体外能有效抑制HepG2细胞VEGF基因表达.
AIM： To construct vascular endothelial growth factor （VEGF） siRNA expression vector and to evaluate its inhibitory cells. effect on VEGF in HepG2 METHODS： Sequencer of a short hairpin RNA targeting VEGF of HepG2 cells was designed and synthesized, pSUPER-VEGF-siRNA was constructed and transfected to HepG2 cells by lipofectamine 2000. The expression of VEGF protein in HepG2 cells transfected with pSUPER- VEGF-siRNA was detected using RT-PCR and Western blot. RESULTS： After evaluation and sequencing, pSUPER-VEGF-siRNA expression vector was successfully constructed. The expressions of VEGF mRNA and VEGF165 protein were decreased markedly after pSUPER-VEGF-siRNA transfection using lipofectamine 2000 and the inhibitive ratio was 65% and 74%, respectively; expressions of VEGF mRNA was lower in experimental group than negative control group and empty vector group （0.304 ± 0.062 vs 0.896 ± 0.061, 0.884 ± 0.074, P 〈 0.05）. CONCLUSION： VEGF siRNA expression vector （pSUPER-VEGF-siRNA） is successfully constructed which can significantly inhibit VEGF gene expression in HepG2 cells.
出处
《世界华人消化杂志》
CAS
北大核心
2009年第2期186-189,共4页
World Chinese Journal of Digestology
基金
湖南省自然科学基金资助项目
No.05JJ30041
湖南省卫生厅科研基金资助项目
No.B2005101
湖南省高等学校科学研究项目
No.05C468~~
关键词
RNA干扰
血管内皮细胞生长因子
肝癌
RNA interference
Vascular endothelial growth factor
Hepatocarcinoma
