靶向沉默大鼠星形胶质细胞VEGF表达siRNA的筛选  

Screening of siRNAs targetting to silence expression of vascular endothelial growth factor in astrocytes of rat

作　　者：邓志宽[1] 吴静[1] 程赛宇[1] 赵士福[1] 李黔宁[1] 

机构地区：[1]第三军医大学新桥医院神经内科,重庆400037

出　　处：《重庆医学》2009年第21期2688-2690,2694,共4页Chongqing medicine

基　　金：第三军医大学中青年基金资助项目(2003D090)

摘　　要：目的筛选靶向沉默大鼠星形胶质细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的短链干扰RNA(short interfering RNA,siRNA)。方法建立大鼠星形胶质细胞培养模型,根据siRNA原理设计、构建、合成、纯化和定量4种靶向大鼠VEGF的siRNA(T1,T2,T3,T4),应用琼脂糖凝胶电泳观察其纯度。将合成的4种siRNA转染至星形胶质细胞,应用实时荧光定量RT-PCR检测星形胶质细胞VEGF mRNA含量,应用免疫细胞化学及Western蛋白印迹法半定量星形胶质细胞VEGF的表达。结果合成的4种siRNA纯度高。转染T1、T2、T3后,培养的星形胶质细胞VEGF mRNA含量、VEGF表达与对照组相比差异无统计学意义(P>0.05)。而转染T4 siRNA的星形胶质细胞其VEGF mRNA、VEGF的表达与对照组相比显著下降(P<0.01)。结论筛选出的T4 siRNA对培养的星形胶质细胞VEGF的表达起特异性抑制作用,其机制与T4siRNA使VEGFmRNA的含量减少有关。Objective To screen short interfering RNAs（siRNA） targetting to silence the expression of vascular endothelial growth factor （VEGF） in astrocytes of rat. Methods Astrocyte culture model of rat was established. Design,construction,synthesis, purification, quantitation of 4 siRNAs（T1, T2, T3, T4） were according to priciples of siRNA. Agarose gel electrophoresis was used to identify the purification of constructed 4 siRNAs. Then 4 siRNAs were transfected respectively to cultured astrocytes. The cultured cells were collected and processed for immunocytochemistry to detect the expression of VEGF, for realtime quantitative RT PCR to detect expression of VEGF mRNA,as well as for Western blots to detect VEGF expression. Results Each of 4 constructed siRNAs had a high degree of purity. After transfection with T1 ,T2,T3 siRNA respectively,the content of VEGF mRNA and the expression of VEGF in cultured astrocytes was not changed significantly when compared with that in control group（all P〉 0.05）. While after transfection with T4 siRNA,which changed significantly（P〈0.01 respectively）. Conclusion T4 siRNA may silence the expression of VEGF in cultured astrocyted of rat by diminishing VEGF mRNA in astrocytes.

关 键 词：SIRNA 血管内皮生长因子 星形胶质细胞 

分 类 号：R363[医药卫生—病理学]