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

doi:10.3969/j.issn.1006-7795.2017.04.012

首都医科大学学报 ›› 2017, Vol. 38 ›› Issue (4): 546-552.doi: 10.3969/j.issn.1006-7795.2017.04.012

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

陈雪莹¹, 姚烨¹, 周高适¹, 陶诗婉¹, 陈兆煜², 谈智^3,4

1. 中山大学中山医学院临床医学系, 广州 510080;
2. 中山大学附属第三医院神经内科, 广州 510630;
3. 中山大学中山医学院高血压研究所, 广州 510080;
4. 中山大学中山医学院生理教研室, 广州 510080

收稿日期:2016-11-21 出版日期:2017-07-21 发布日期:2017-07-20
通讯作者: 谈智 E-mail:tanzhi@mail.sysu.edu.cn
基金资助:
国家自然科学基金（81270377），广东省科技厅社会发展领域项目（2015A020212020），广东省自然科学基金项目（2014A030313062）

Effect of vascular injury on the expression of polymerase Ⅰ and transcript release factor in vascular smooth muscle and its mechanism

Chen Xueying¹, Yao Ye¹, Zhou Gaoshi¹, Tao Shiwan¹, Chen Zhaoyu², Tan Zhi^3,4

1. Department of Clinical Medicine, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China;
2. Department of Neurology, The 3rd Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510630, China;
3. Institute of Hypertension, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China;
4. Department of Physiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China

Received:2016-11-21 Online:2017-07-21 Published:2017-07-20
Supported by:
This study was supported by National Natural Science Foundation of China (81270377), Science and Technology Department of Guangdong (2015A020212020), Natural Science Foundation of Guangdong (2014A030313062)

摘要/Abstract

摘要： 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。

关键词: 血管损伤, 平滑肌细胞, 泛素化, 血管再狭窄

Abstract: 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.

Key words: vascular injury, smooth muscle cell, ubiquitination, vascular restenosis

中图分类号:

陈雪莹, 姚烨, 周高适, 陶诗婉, 陈兆煜, 谈智. 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[J]. 首都医科大学学报, 2017, 38(4): 546-552.

Chen Xueying, Yao Ye, Zhou Gaoshi, Tao Shiwan, Chen Zhaoyu, Tan Zhi. Effect of vascular injury on the expression of polymerase Ⅰ and transcript release factor in vascular smooth muscle and its mechanism[J]. Journal of Capital Medical University, 2017, 38(4): 546-552.

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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

Effect of vascular injury on the expression of polymerase Ⅰ and transcript release factor in vascular smooth muscle and its mechanism

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