核转录因子NF-κB对血管损伤和新生内膜形成的影响

Role of the antisense and decoy oligonucleotide of NF-κB in the vessel injury and neointimal formation

目的:探讨核转录因子NF-κB对血管平滑肌细胞增殖以及大鼠颈动脉球囊损伤后血管新生内膜的作用。方法:原代培养大鼠胸主动脉血管平滑肌细胞。检测增殖的平滑肌细胞内增殖细胞核抗原(PCNA)和NF-κB水平。制作大鼠血管球囊损伤模型,检测血管新生内膜形成及单核细胞化学趋化因子(MCP-1)、NF-κBp65和细胞外信号调节激酶(ERK2)的表达。结果:增殖的平滑肌细胞PCNA和NF-κBp65蛋白水平表达增加。NF-κBp65反义和诱骗寡核苷酸抑制PCNA表达。大鼠血管球囊损伤后第7天,正义组、诱骗对照组、模型组的内膜面积、中膜面积、内膜/中膜比值达到高峰。反义组、诱骗组和反义诱骗组显著降低内膜与中膜比值(P<0.05)。球囊损伤后3d、5d、7d,MCP-1mRNA和蛋白质水平持续而明显的表达增强,14d后略为降低。反义组、诱骗组、反义诱骗组在各时间点均能减少MCP-1mRNA和蛋白质表达。Western Blot检测显示血管球囊损伤后7d,NF-κBp65、ERK2的蛋白合成达到高峰。反义组、诱骗组、反义诱骗组较模型组、正义组、诱骗对照组各时相点蛋白合成均减弱。结论:增殖的平滑肌细胞NF-κBp65基因表达增加。NF-κB调控PCNA、MCP-1、ERK2的基因表达和蛋白质水平。局部转染NF-κB反义和诱骗寡核苷酸能抑制血管新生内膜的形成。

Objective:To investigate the effect of local orientation of antisense and decoy oligonucleotide of nuclear factor kappa B on restenosis after angioplasty and smooth muscle cell proliferation.Method:Rat aortic smooth muscle cells were isolated from male Sprague-Dawley rats thoracic aorta.Proliferating cell nuclear antigen(PCNA)protein synthesis was detected in proliferating smooth muscle cell.NF-κB protein level was measured in proliferating smooth muscle cells.Sprague-Dawley rats underwent balloon-dilation injury of the left carotid artery,then we examined the in vivo effect of the antisense or/and decoy oligonucleotides of NF-κB on intima proliferation and MCP-1 、NF-κBp65 and ERK2 expression.Result:PCNA protein synthesis was increased in proliferating smooth muscle cell.NF-κB p65 protein level was increased in proliferating smooth muscle cells.PCNA expression was inhibited in proliferating smooth muscle cell by the use of antisense and decoy oligonucleotides.In model group,sense group and scramble group,vessel intima area,media area and intima/media ratio reached the maximum at 7 days after injury.Antisense group,decoy group and antisense plus decoy group improved these observational index(P<0.05).MCP-1 mRNA and protein expression were maximal at 7 days after balloon injury.Compared with model group,sense group and scramble group,antisense group,decoy group and antisense plus decoy group all had lowered MCP-1 mRNA and protein expression in every time point(P<0.05).Western blot analysis showed NF-κB p65 and ERK2 protein synthesis reached the peak at 7 days,while protein expression after 14 days was similar to that of 7 days.In antisense group,decoy group and antisense plus decoy group,protein synthesis was inhibited more significantly than those of model group,sense group and scramble group(P<0.05).Conclusion:NF-κB expression was increased in proliferating smooth muscle cells.NF-κB may modulate genes expression and protein synthesis of PCNA,MCP-1 and ERK2.Antisense and decoy oligonucleotide of NF-κB by local lipofectamine transfer would inhibit vascular neointima formation.