转录因子NF-κB对血管成形术后血管狭窄和新生内膜形成的影响

Role of the transcription factor NF-κB in the vessel stenosis and neointima formation in balloon-injured rat artery

目的探讨核转录因子NF-κB的反义和诱骗性寡核苷酸对大鼠颈动脉球囊损伤后血管增殖反应及相应细胞因子、细胞外信号调节激酶的影响。方法SD大鼠随机分为7组,每组分为6个时相点(6 h,1,3,5,7,14 d),每个时相点3只大鼠。制作血管球囊损伤模型。相应时间点处死动物,使用病理形态学、逆转录聚合酶链反应、免疫组化、W esternb lot等方法测定血管病理形态改变、单核细胞趋化因子(Monocytes chemotactic prote in-1,MCP-1)的mRNA和蛋白质表达、NF-κB p65和细胞外信号调节激酶(Extra-cellu lar sig-nal regu lated k inase,ERK2)的蛋白质含量。结果模型组、正义组、Scramb le组血管内膜/中膜在d 5增加,14 d达到高峰。反义组、诱骗组、反义组+诱骗组干预后,血管内膜/中膜明显减小(P<0.05),反义组+诱骗组较反义组、诱骗组治疗效果不明显。MCP-1 mRNA表达在血管损伤后6 h即可检测到,3、5、7 d后持续表达增加,14 d后表达降低,免疫组化显示MCP-1蛋白质表达在6个时相点均为阳性染色,14 d达到高峰;反义组、诱骗组、反义组+诱骗组治疗后,与模型组、正义组、Scramb le组相比,MCP-1 mRNA表达和蛋白合成在各时相点均降低(P<0.05)。W estern b lot检测核蛋白抽提物显示核因子NF-κB p65在血管损伤后6 h有一定蛋白表达,1 d后蛋白表达明显增加,至7 d达高峰,14 d后蛋白表达略降低。ERK2在血管损伤后1 d蛋白表达开始增加,d 7达到峰值,14 d后与d 7相比无明显差异。反义组、诱骗组、反义组+诱骗组干预后,各时相点蛋白质表达均较模型组、正义组、Scramb le组减弱(P<0.05)。结论转录因子NF-κB调控MCP-1、ERK2基因表达和蛋白质合成;球囊损伤后血管壁细胞增殖在不同时相点有动态变化;脂质体介导局部转染反义、诱骗性寡核苷酸可抑制NF-κB激活对下游基因的调控,两者联合作用效果较单独应用未表现出更明显的作用。

Aim To examine the in vivo effect of the antisense or/and decoy oligonucleotide of NF-KB on vessel proliferation and balloon-injured monocytes chemotactic protein-1 （ MCP-1） and extracellular signal regulated kinase in the carotid artery of rats. Methods Sprague-Dawley rats underwent balloon-dilation injury of the left carotid artery. The rats were divided into 7 groups（n = 18 ） and each group were further divided into 6 sub-groups＇for study at 6 time points （1,3,5,7,14 days,n =3）. Uninjured artery of the same rat was used as the control. Results In model group, sense group and scramble group, intima/media ratio increased after 5 days and reached the maximum after 14 days; intima/media ratio in antisense group, decoy group, antisense plus decoy group decreased significantly （P 〈 0.05 ）. The effects on antisense plus decoy group were not obvious as that of antisense group and decoy group alone. MCP-1 mRNA expression was examined after 6 hours of artery injury, but not evident after 1 day. It was increased continuously after 3,5 and 7 days and decreased after 14 days. Comparing with model group, sense group and scramble group, antisense group, decoy group and antisense plus decoy group all had lowered MCP-1 mRNA expression in every time point （ P 〈 0.05 ）. Immunohistochemistry studies revealed that MCP-1 protein as positively stained at the six time points and the protein expression reached its maximal after 14 days. In antisense group, decoy group and antisense plus decoy group, MCP-1 protein synthesis decreased at every time point comparing with the model group, sense group and scramble group. Western blot studies showed NF-kB p65 was positively stained after 6 hours of injury and increased after 1 day and reached its peak after 7 days, but protein expression was decreased after 14 days. ERK2 protein synthesis increased after 1 day and reached its peak after 7 days, while protein expression after 14 days was similar to that of 7 days. antisense group, decoy group and antisense plus decoy group treatment inhibited protein synthesis more significantly than that of model group, sense group and scramble group （P 〈 0.05）. Conclusions Nuclear factor NF-kB modulated genes expression and protein synthesis of MCP-1 and ERK2;celluar proliferation in vessel wall dynamically changed after balloon angioplasty injury; antisense and decoy oligonucleotide of NF-kB transfered by local lipofectamine inhibited NF-kB activating genes modulation and the combined effects were not more remarkable than that of single treatment.