构建siRNA慢病毒载体抑制A549细胞株VEGF受体KDR基因的表达  

Inhibition of VEGF Receptor KDR Gene Expression in A549 Cell Line by Lentivirus-Mediated siRNA

作　　者：王彤[1] 李丹妮[1] 李咏[1] 王杰[1] 

机构地区：[1]沈阳医学院生物化学教研室,辽宁沈阳110034

出　　处：《临床医学工程》2012年第10期1664-1666,共3页Clinical Medicine & Engineering

基　　金：辽宁省教育厅科研基金资助项目(2009B176);沈阳医学院科研基金资助项目(20101020)

摘　　要：目的构建针对血管内皮生长因子(VEGF)受体KDR基因的siRNA慢病毒载体,鉴定其对肺癌细胞株A549基因干扰效果。方法设计合成针对KDR编码区的三条siRNA序列及阴性对照序列,克隆到PGCL-GFP载体,构建重组质粒,行PCR鉴定、DNA测序分析;转染人肺癌细胞株A549,Real-Time PCR及Western blot分别检测KDR mRNA、蛋白水平变化;细胞计数法绘制细胞生长曲线,流式细胞仪检测细胞周期。结果成功构建pGCL/KDR-siRNA重组质粒。A549干扰组KDR mRNA表达率下降,KDR蛋白表达量明显减少,细胞周期改变。结论构建的pGCL/KDR-siRNA表达载体可在mRNA和蛋白水平上有效抑制A549细胞株KDR基因的表达。Objective To construct the siRNA vector targeting human vascular endothelial growth factor （VEGF） receptor KDR and to detect its silencing effects on human lung cancer cell line A549. Methods Three siRNA sequences and one negative control sequence were designed for the KDR encoding region, and then cloned into PGCL-GFP vector. PCR and DNA sequencing were used to testify recombinant plasmids. Then the expression vectors were transferred into A549 cell line to produce packaged lentivirus. Expresssion of rnRNA and protein in A549 cell line were detected by Real-Time PCR and Western blot. Cell counting and FCM were used to explore cell growth. Results PGCL-GFP vector were constructed successfully. KDR expression of A549 cells in test group dropped and KDR protein decrease, as well as cell number decrease and cell cycles change. Conclusions The constructed pGCL/KDR-siRNA vector effectively inhibits the expression ofKDR gene in A549 cell line by mRNA and protein levels.

关 键 词：KDR RNA干扰 慢病毒 A549细胞株 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]