血管损伤反应对血管平滑肌合酶Ⅰ和转录释放因子表达的影响及机制

摘要

Objective To investigate the role of polymerase Ⅰ and transcript release factor or Cavin-1 in vascular restenosis after vascular injury and its molecular mechanism.Methods 200-220 g Sprague Dawley(SD)male rats were randomly divided into sham and injury groups with injury groups establishing balloon injury models.Cellular content from carotid taken after the sham and balloon injuries were shown by hematoxylin and eosin staining.Expression Cavin-1 protein from above groups were determined by Western blotting and immunofluorescence.The Cavin-1 mRNA from above groups was detected by real-time RT-PCR, respectively.Immunochemical staining, Western blotting and Co-immunoprecipitation revealed ubiquitinated protein in carotid artery from sham and injury groups.Rat aortic smooth muscle cells were divided into: ①control group(CTRL);②CHX group rats were pretreated with cycloheximide (CHX, 25μmol/L) for 1 h;③CHX+MG group rats were pretreated with CHX (25μmol/L) for 1 h followed by treatment with MG 132(10 μmol/L)for additional 24 hours.④CHX+CQ group rats were pretreated with CHX (25μmol/L) for 1 h followed by treatment with chloroquin(CQ,50 μmol/L)for additional 24 hours.Expression Cavin-1 protein from above groups were determined by Western blotting.Results As for animal models, the carotid vascular wall became thicker after the balloon injury and the expression of Cavin-1 protein was significantly decreased(P<0.05) while real-time RT-PCR showed no significant difference of Cavin-1 mRNA between sham and injury groups.Ubiquitinated protein levels were higher in the injured carotid compared with sham group(P<0.05) As for cell experiment, expression of Cavin-1 was decreased significantly from CHX group compared with CTRL(P<0.05).CHX+MG group can reverse the above effects (P<0.05).Conclusion Expression of Cavin-1 protein from balloon injury carotid arteries were reduced, and its mechanism may relate to up-regulated ubiquitination degradation pathway.%目的 探讨聚合酶Ⅰ和转录释放因子(polymerase Ⅰ and transcript release factor,PTRF or Cavin-1)在损伤血管后血管再狭窄病理过程的作用及分子机制.方法 将200~220 g雄性Sprague Dawley大鼠采用数字表法随机分为假手术组和血管损伤组,血管损伤组制备颈动脉损伤动物模型.用苏木精伊红染色显示损伤后颈动脉的结构,免疫荧光、Western blotting检测损伤后血管的Cavin-1蛋白表达,real-time RT-PCR方法检测损伤后血管的Cavin-1 mRNA表达,免疫组织化学、Western blotting、免疫共沉淀法检测颈动脉泛素化蛋白的表达.大鼠动脉平滑肌细胞实验分为①空白对照组(control,CTRL);②CHX组:用放线菌酮(cycloheximide, CHX,25 μmol/L)预处理1 h;③CHX+MG组:用CHX预处理1 h后,接着用MG 132(蛋白酶体抑制剂,10 μmol/L)处理24 h;④CHX+CQ组:用CHX预处理1 h后,接着用氯喹(chloroquin,CQ,10 μmol/L)处理24 h.应用Western blotting检测细胞Cavin-1蛋白浓度.结果 动物模型中,球囊损伤后,血管壁明显增厚,Cavin-1表达显著下降(P<0.05),Cavin-1 mRNA表达差异不明显,泛素化蛋白表达显著上调(P<0.05).细胞实验中,应用CHX预处理可明显降低平滑肌细胞Cavin-1的表达(P<0.05),而CHX+MG可以明显对抗CHX上述作用过程(P<0.05).结论 球囊损伤颈动脉后,血管的Cavin-1蛋白表达下调,其机制可能与增加的泛素化-蛋白酶体降解途径有关.