VEGF靶向性siRNA表达载体的构建及其沉寂作用

Vector construction and silencing effect of VEGF gene targeted small interfering RNA

目的:构建针对VEGF的siRNA表达载体,转染入人骨肉瘤细胞系MG-63观察其对细胞VEGF表达的沉默效应.方法:设计VEGF发卡样siRNA,依据设计合成两条互补的寡核苷酸链,退火后连接入T载体,转化扩增后进行序列测定.用脂质体包裹转染骨肉瘤细胞MG-63,用RT-PCR方法检测细胞VEGF mRNA表达水平.结果:把针对VEGF基因的siRNA的双链寡合苷酸片断克隆到表达载体pSuperneo,经过酶切鉴定与测序,结果正确;稳定转染人骨肉瘤细胞系MG-63后,用RT-PCR方法检测细胞VEGF mRNA表达水平明显降低.结论:成功构建出针对VEGF的siRNA表达载体pSuper-neo-VEGF,转染骨肉瘤细胞后能显著降低细胞VEGF mRNA表达.

AIM：To construct VEGF gene-targeted small interfering RNA（siRNA） and its expression vector; and to observe the expression level of its expression vector VEGF gene in human osteosarcoma MG-63 cell transfected by the vector. METHODS：First to design VEGF gene-targeted hairpin siRNA,then to synthesize two complementary oligo nucleotide strand,after annealing,to insert the two-strand oligo nucleotide into pSuper neo vetor,which was then restrictive enzyme digested and sequenced. After that we transfected human osteosarcoma MG-63 cell line with the vector using lipofeetamine method. Finally, detected the mRNA expression level of VEGF gene through RT- PCR. RESULTS： Restrictive enzyme digestion and sequencing confirmed the vector containing siRNA was what we wanted; RT- PCR showed the expression level of VEGF gene in transfected MG-63 was decreased. CONCLUSION： We had constructed the correct VEGF gene-targeted siRNA and its vector, and the vector could obviously reduce the VEGF gene expression after transfecting.