RNA干扰技术沉默Smad4基因表达对ECV304细胞增殖和迁移的影响  

Influence of RNA interference-induced silencing Smad 4 gene on proliferation and migration of ECV304 cell line

作　　者：曹阳[1] 黄晓园[2] 马全富[2] 杨漾[1] 庄亮[2] 马丁[2] 周剑峰[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院血液内科,武汉430030 [2]华中科技大学同济医学院附属同济医院妇产科,武汉430030

出　　处：《肿瘤》2008年第12期1019-1022,共4页Tumor

基　　金："九七三"国家重点基础研究发展计划项目(编号:2002CB513100)

摘　　要：目的:探讨利用RNA干扰技术下调Smad 4基因表达对人脐静脉内皮细胞增殖和迁移能力的影响,初步了解Smad 4在血管生成中的作用。方法:设计并合成靶向人Smad4基因的小分子干扰RNA(small interfering RNA,siRNA)片段siRNA1和siRNA2,脂质体介导转染入人脐静脉内皮细胞株ECV304。应用实时定量RT-PCR和Western印迹法检测转染前后Smad4基因表达水平;应用细胞周期分析和羧乙基锗倍半氧化物(6-carboxyfluorescein diacetate succinimidyl ester,CFSE)-流式细胞仪检测ECV304细胞转染前后细胞增殖能力的变化;通过单层细胞划痕实验观察细胞转染前后迁移能力的变化。结果:从2条siRNA中成功筛选出1条siRNA,于RNA和蛋白水平可明显下调Smad4基因的表达;ECV304细胞转染Smad4的siRNA后,细胞的增殖和迁移能力明显增强。结论:利用RNA干扰技术能够筛选出高效的特异阻断Smad4基因表达的siRNA;Smad4基因表达下调能够明显促进血管内皮细胞的增殖和迁移。Objective： To investigate the effect of silencing Smad4 gene expression by using small interfering RNA（siRNA） on proliferation and migration of ECV304 cell line and explore the role of Smad4 in angiogenesis. Methods： Two siRNA sequences targeting Smad4 gene including siRNA1 and siRNA2 were designed and then synthetized. The two sequences of siRNA were transfected into ECV304 cells via lipofectamine mediation. The alteration of Smad4 mRNA and protein expression was examined by real-time RT-PCR and Western blot, respectively. The proliferation capacity of ECV304 ceils was measured by cell cycle analysis and 6-carboxyfluorescein diacetate succinimidyl ester （CFSE） flow cytometry. The monnstratal wound healing test was used to determine the migration capacity of ECV304 cells. Results： siRNA2 was successfully screened out of the two sequences. Transfection of siRNA2 significantly knocked down the Smad4 mRNA and protein expressions. The proliferation and migration capacity of ECV304 cells are significantly enhanced after transfection with siRNA2. Conclusions： Highly effective and specific siRNA targeting Smad4 gene can be successfully screened by RNA interference technique. Selective silencing of Smad 4 gene greatly promotes the proliferation and migration of ECV304 cells.

关 键 词：肿瘤 实验性 RNA干扰 细胞增殖 基因表达 基因 SMAD4 细胞 ECV304 

分 类 号：R730.23[医药卫生—肿瘤]