【摘要】 目的探讨Grb2的抑制对K562细胞侵袭和迁移能力的影响。方法 pSilence-4.1CMV-Grb2原核表达载体的构建并应用脂质体介导法将其转染至人慢性髓细胞性白血病细胞K562,后以潮霉素筛选稳定表达siRNA的细胞克隆。应用Western blot和RT-PCR技术,检测转染Grb2siRNA重组质粒的K562细胞(K562/pSilence-4.1CMV-Grb2)、转染空载体细胞(K562/pSilence-4.1CMV)和未转染细胞(K562)中Grb2的表达情况。Transwell小室体外迁移和侵袭能力实验测定各组细胞的迁移和侵袭能力。结果成功构建了pSilence-4.1CMV-Grb2原核表达载体,并获得稳定表达siRNA原核载体的细胞株K562/pSilence-4.1CMV-Grb2/405、K562/pSilence-4.1CMV-Grb2/541及K562/pSilence-4.1CMV-Grb2/215;与其他组比较,K562/pSilence-4.1CMV-Grb2/405细胞Grb2mRNA和蛋白的表达水平显著降低并伴随着其迁移和侵袭能力的下降。结论 Grb2的抑制可明显降低K562细胞侵袭和迁移能力。提示针对Grb2的siRNA有望成为抑制肿瘤转移的基因治疗的一种新途径。更多还原

【Abstract】 Objective To study the effect of inhibition of Grb2 expression level on K562 cell invasion and migration by RNA interference.Methods Eukaryotic expression vector pSilence-4.1CMV-Grb2 was constructed and stably transfected into chronic myelognous leukemia cells K562 using lipofectamine,K562/pSilence-4.1CMV-Grb2 cell lines stably expressing Grb2 siRNA were established with hygromycin selection.The downregulation of Grb2 by stable expression of Grb2 siRNA was confirmed by Western blot and RT-PCR.Cell migration and invasion was monitored by transwell assay.Results Three cell lines with Grb2 siRNA stable expression were established,including K562/pSilence-4.1CMV-Grb2/405,K562/pSilence-4.1CMV-Grb2/541 and K562/pSilence-4.1CMV-Grb2/215.Grb2 mRNA and protein expression levels in K562/pSilence-4.1CMV-Grb2/405 cells were down-regulated and a decrease in cell migration and invasion was observed in these cells.Conclusions Downregulation of Grb2 in K562 cells could lead to a decrease in cell migration and invasion.Our results suggest that gene therapy targeting Grb2 by siRNA may inhibit tumor metastasis.更多还原