RNAi沉默喉鳞癌Hep-2细胞株VEGF-C mRNA基因表达的实验研究  

Silencing Effects of RNAi on VEGF-C mRNA Expression in Hep-2 Cells

作　　者：路武豪[1] 娄卫华[1] 赵国强[2] 

机构地区：[1]郑州大学第一附属医院耳鼻咽喉科,河南郑州450052 [2]郑州大学基础医学院微生物与免疫学教研室,河南郑州450052

出　　处：《肿瘤基础与临床》2008年第2期99-102,共4页journal of basic and clinical oncology

摘　　要：目的构建针对喉鳞癌细胞株Hep-2中血管内皮生长因子C(vascular endothelial growthfactor C,VEGF-C)的siRNA表达载体,研究载体介导的RNAi技术对喉鳞癌细胞株Hep-2中VEGF-C mRNA表达的抑制作用。方法依据siRNA靶序列的设计原则,以VEGF-C为靶基因,同时随机组合出一条对照序列,分别合成2对编码短发卡结构的两条DNA序列,经退火合成互补DNA双链,克隆入siRNA表达载体pSuper-neo中,转化鉴定后进行PCR和DNA序列测定。用脂质体介导转染入喉鳞癌Hep-2细胞株,采用RT-PCR方法检测细胞VEGF-C基因mRNA水平的变化。结果成功构建发卡样VEGF-C siRNA真核表达载体;转染VEGF-C靶向siRNA(pSuper-neo-siV1)的Hep-2细胞,VEGF-C基因的表达水平较无关对照组pSuper-neo-siV2和转染空载体pSuper-neo的对照组显著下降。结论成功构建载体介导的VEGF-C靶向RNA干扰重组体,并能有效抑制Hep-2细胞中VEGF-C mRNA表达。Objective To construct the expression vector of VEGF-C and to investigate the effects of RNAi on VEGF-C mRNA expression in Hep-2 cells. Methods According to the VEGF-C mRNA sequence,GenBank had been adopted,we used the siRNA design and analyzed software to design the target oligonueleotides. Afterwards,the two siRNA oligonueleotides of VEGF-C mRNA were annealed. The pSuper-neo- VEGF-C siRNA expression vector was constructed by gene recombination,then transfeeted into the cultured Hep-2 cells by liposome method. The positive cell clones were screened with G418. The stable transfeetion and expression of VEGF-C mRNA in Hep-2 were determined by RT-PCR. The pSuper-neo transfeeted plasmid was used as control. Results pSuper-neo-VEGF-C siRNA expression vector was successfully constructed. Liposome-meditated gene trandfeetion of pSuper-neo-VEGF-C siRNA expression vector into Hep-2 cell down-regulated the mRNA expression level of VEGF gene,as compared with the control group. Conclusions The pSuper-neo-VEGF-C siRNA expression vector can inhibit the expression of VEGF-C mRNA in Hep-2 cells.

关 键 词：喉鳞癌 VEGF—C RNA干扰 HEP-2细胞株 

分 类 号：R739.65[医药卫生—肿瘤]