CBP基因沉默对大鼠颈动脉球囊损伤后内膜增生的影响

Effect of CREB Binding Protein Silencing on Neointimal Hyperplasia in Rat Carotid Arteries After Balloon Injury

目的观察小干扰RNA(siRNA)重组慢病毒介导的CREB结合蛋白(CBP)基因沉默对大鼠颈动脉球囊损伤后新生内膜增生的影响,并探讨其作用机制。方法雄性SD大鼠48只,随机分为4组:假手术组、PBS对照组、慢病毒介导CBP基因siRNA转染组(CBP-siRNA-Lenti组)及慢病毒介导非CBP同源序列siRNA转染组(NC-siRNA-Lenti组),建立大鼠颈动脉球囊损伤模型,术后28天处死动物。分别用实时定量PCR、Western Blot检测大鼠颈动脉CBP和乙酰化核因子κB p65(NF-κB p65)的表达水平;病理组织学观察血管内膜增生情况;免疫组织化学染色对损伤血管壁增殖细胞核抗原(PCNA)的表达进行评估。结果术后28天,与PBS对照组和NC-siRNA-Lenti组比较,CBP-siRNA-Lenti组CBP mRNA和蛋白的表达显著下调(P均<0.05),CBP沉默能明显抑制新生内膜面积(0.108±0.008 mm2比0.238±0.022 mm2、0.252±0.016 mm2,P<0.05)、内膜与中膜面积比(0.706±0.062比1.483±0.136、1.497±0.137,P<0.05)的增加,及下调血管壁乙酰化NF-κB p65和PCNA的表达水平(P均<0.05)。结论慢病毒介导的CBP基因沉默能有效地抑制颈动脉球囊损伤后新生内膜的形成,其机制可能与抑制NF-κB p65的过度乙酰化有关。

Aim To investigate the effects of siRNA recombinant lentivirus targeting CREB binding protein （CBP） gene on neointimal hyperplasia in rat carotid arteries after balloon injury and its possible mechanism. Methods Forty-eight male SD rats were randomly divided into four groups： Sham group, PBS group, CBP-siRNA-Lentivirus group and NC-siRNA-Lentivirus group to establish the model of balloon-injury in left common carotid artery, and the rats were sacrificed 28 days after injury and in vivo gene transfer. The expression of CBP and acetylated nuclear factor kappaB p65 （NF-KB p65） were determined by real-time PCR and Western Blot. Proliferating cell nuclear antigen （PCNA） protein expression was detected by immunohistochemistry. Meanwhile, morphometric analysis was used to measure neointimal hyperplasia. Results 28 days after operations, lentivirus siRNA targeting CBP markedly decreased CBP mRNA and protein expression compared with PBS and NC-siRNA-Lentivirus groups （ P 〈 0. 05 ）. CBP gene silencing significantly reduced the neointimal area （0. 108±0. 008 mm2 vs 0. 238 ±0. 022 mm2 and 0. 252 ±0. 016 mm2, P 〈0. 05） and intima / media ratio （0. 706 ± 0. 062 vs 1. 483 ± 0. 136 and 1. 497 ± 0. 137, P 〈 0. 05 ）. Furthermore, compared with PBS and NC-siRNA-Lentivirus groups, PCNA and acetylation of NF-κB p65 expression were both obviously downregulated in CBP-siRNA-Lentivirus group （P 〈 0. 05 ）. Conclusions Lentivirus-mediated CBP gene silencing could efficiently suppress neointimal formation in balloon injured rat carotid artery, and the mechanism was involved with downregulation of NF-κB p65 acetylation.