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
李玲 1王风华 2连鸿凯 3白玉 3蔡朝阳 3赵国强 4马俊芬4
作者信息
- 1. 郑州市骨科医院,郑州大学,河南,郑州,450052;郑州大学基础医学院,河南,郑州,450052
- 2. 郑州大学第五附属医院,河南,郑州,450052
- 3. 郑州市骨科医院,郑州大学,河南,郑州,450052
- 4. 郑州大学基础医学院,河南,郑州,450052
- 折叠
摘要
目的:构建针对VEGF的siRNA表达载体,转染人人骨肉瘤细胞系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pSuperneo-VEGF,转染骨肉瘤细胞后能显著降低细胞VEGF mRNA表达.
Abstract
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 lipofectamine method.Finally,detected the mRNA expression level of VEGF gene through RTPCR.RESULTS:Restrictive enzyme digestion and sequencing confirmed the vector containing siRNA was what we wanted;RTPCR 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.
关键词
RAN干扰/血管内皮生长因子/骨肉瘤Key words
siRNA/VEGF/osteosarma引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据