NF-κB p65 siRNA抑制人脐静脉内皮细胞迁移的研究

Small interfering RNA targeting NF-κB Inhibits human umbilical venous endoth-elial cells migration

目的:观察RNA干扰技术(RNAi)特异性抑制人脐带静脉血管内皮细胞(EAhy926)内NF-κBp65基因效果及对细胞迁移力的影响。方法:10μg/L血管内皮生长因子(VEGF)刺激培养EAhy926细胞,给予转染NF-κBp65siR-NA,同时设立对照组,分别是阴性对照siRNA转染组、空白脂质体组及正常对照组(不给予其他任何处理),48h后分别采用RT-PCR、Western blot检测细胞内NF-κBp65mRNA及蛋白的表达水平,并计算细胞迁移抑制率。结果:与正常对照组比较,NF-κBp65siRNA组的细胞内NF-κBp65mRNA及蛋白的表达量均明显减少(P<0.05);而阴性对照siRNA转染组及空白脂质体组的细胞内相应的表达量则无统计学意义。同时,siRNA转染后对细胞的迁移抑制率达64.5%,空白脂质体组和阴性对照siRNA组则仅为7.5%和11.3%。结论:siR-NA能通过抑制血管内皮细胞内NF-κBp65的信号通路,抑制细胞的迁移能力,这为今后抗血管生成治疗研究打下了基础。

AIM： To investigate the effect of small interfering RNA（siRNA） targeting NF-κB on migration ability of human umbilical venous endothelial cells. METHODS： EAhy926 cells （ Human umbilical vein endothelial cell line） were cultured with stimulation of vascular endothelial growth factor （ VEGF）, and then were transfected with NF-vB -siRNA （40 pmol/L） by using catiotic liposome lipofectamineTM 2000 as the transfecting agent. Cells transfected with negative control-siRNA （40 pmol/L）, empty Liposome and nothing as control. Forty-eight hours later, cells were collected and prepared for RT-PCR, Western blot analysis, and determination of the migration inhibition rate. RESULTS： Compared with normal control group, both mRNA and protein levels of NF-κB were decreased obviously and at the same time cell migration inhibition rate was increased obviously in NF-κB- siRNA group. There were no significant differences between empty liposome group, negative control siRNA group and normal control group, respectively （ P 〉 0.05）. CONCLUSION： Synthetic siRNA can effectively inhibit endothelial cell migration ability by means of blocking the signaling pathway of NF-κB. And siRNA targeting NF-κB may be useful for antiangiogenesis treatment.