中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (37): 6942-6946.doi: 10.3969/j.issn.1673-8225.2011.37.024

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

E1A激活基因阻遏子蛋白过表达可抑制人血管平滑肌细胞的迁移

闫承慧，栾  波，李  杰，张效林，韩雅玲   

  1. 解放军沈阳军区总医院全军心血管病研究所心内科  110840
  • 收稿日期:2011-04-05 修回日期:2011-07-18 出版日期:2011-09-10 发布日期:2011-09-10
  • 通讯作者: 韩雅玲，博士，教授，博士生导师，沈阳军区总医院全军心血管病研究所心内科 110840 hanyaling@ 263.net
  • 作者简介:闫承慧☆，女，1973年生，黑龙江省哈尔滨市人，汉族，2003年哈尔滨医科大学毕业，博士，副主任技师，主要从事心血管病的基础研究。 yanch1029@ 163.com
  • 基金资助:

    课题受由国家自然科学基金面上项目(30770793, 30971218，81070097)，国家自然科学基金青年基金项目(30800465)，辽宁省自然科学基金(20092088)和辽宁省科技攻关项目(2009225009-9)资助。

Overexpression of cellular repressor of E1A-stimulated genes inhibits migration of vascular smooth muscle cells

Yan Cheng-hui, Luan Bo, Li Jie, Zhang Xiao-lin, Han Ya-ling   

  1. Department of Cardiology, Research Institute of Cardiovascular Diseases, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang  110840, Liaoning Province, China
  • Received:2011-04-05 Revised:2011-07-18 Online:2011-09-10 Published:2011-09-10
  • Contact: Han Ya-ling, M.D., Professor, Doctoral supervisor. Department of Cardiology, Research Institute of Cardiovascular Diseases, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang 110840, Liaoning Province, China hanyaling@263.net
  • About author:Yan Cheng-hui☆, Doctor, Associate chief technician, Department of Cardiology, Research Institute of Cardiovascular Diseases, General Hospital of Shenyang Military Area Command of Chinese PLA, Shenyang 110840, Liaoning Province, China yanch1029@ 163.com
  • Supported by:

    the National Natural Science Foundation of China (General Program), No. 30770793*, 30971218*, 81070097*; Youth Foundation Program of the National Natural Science Foundation of China, No. 30800465*; the Natural Science Foundation of Liaoning Province, No. 20092088*; High-Tech Research and Development Program of Liaoning Province, No. 2009225009-9*

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253

被引次数

2

摘要：

背景：成熟动脉中膜血管平滑肌细胞(vascular smooth muscle cells, VSMCs)分化稳态的丧失是血管老化的一个重要原因。
目的：观察E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes，CREG)抑制人血管平滑肌细胞迁移的分子机制。
方法：应用已建立的稳定转染CREG过表达细胞hVSMCs-CREG和CREG表达沉默的hVSMCs-siCREG细胞株通过刮伤实验和Transwell小室细胞移行实验检测细胞迁移能力，通过Western blot检测转染前后细胞中CREG蛋白、基质金属蛋白酶和JNK表达及活化情况，应用JNK和基质金属蛋白酶9抑制剂阻断研究分析上述信号分子表达变化在细胞迁移中的作用。
结果与结论：Western blot结果证实，CREG蛋白表达在hVSMCs-CREG组中增加(P < 0.05)，而在hVSMCs-siCREG组中减少(P < 0.05)。刮伤实验和Transwell实验分析结果证实hVSMCs-CREG组细胞较正常对照组细胞迁移能力受抑。相反，hVSMCs-siCREG组细胞迁移能力显著增加(P< 0.05)。Western blot和明胶酶谱分析证实hVSMCs-siCREG组细胞中MMP9活性明显增加(P < 0.05)，同时JNK蛋白活化。进一步应用JNK抑制剂阻断研究证实，CREG蛋白表达抑制引起的血管平滑肌细胞迁移能力增加与细胞中基质金属蛋白酶9活性均受到剂量依赖性抑制。结果证实，CREG蛋白表达可抑制JNK-基质金属蛋白酶9信号通路的活化，以此抑制体外培养的人血管平滑肌细胞迁移。

关键词: 阻遏蛋白, 腺病毒E1A蛋白, E1A激活基因阻遏子, 血管平滑肌细胞, 迁移, 组织构建

Abstract:

BACKGROUND: Loss of homeostasis in mature vascular smooth muscle cells (VSMCs) plays an important role in senescence of blood vessel. However, the molecular mechanism remains unknown.
OBJECTIVE: To investigate the molecular mechanism by which cellular repressor of E1A-stimulated genes (CREG) inhibits the migration of VSMCs.
METHODS: The pRc/CMV-CREG plasmid or the pSM2-siCREG plasmid was transferred into human VSMCs to produce the cell clone that overexpresses or downregulates CREG respectively. Cell scrape assay and Transwell assay were used to detect the migration of VSMCs. The CREG protein expression, matrix metalloproteinases (MMPs) and JNK expression and activation in cells before and after transfection were detected by western blot analysis. The effect of above-mentioned signal molecule expression on VSMCs migration was analyzed through the use of JNK and MMP9 inhibitor.
RESULTS AND CONCLUSION: Western blot analysis identified that CREG protein expression in hVSMCs-CREG cells increased (P < 0.05), but it was significantly decreased in the hVSMCs-siCREG cells (P < 0.05), compared with control ones. Cell scrape assay and Transwell assay results showed the migration of VSMCs was obviously inhibited in the hVSMCs-CREG group than in the normal control group. However, the migration of VSMCs in the hVSMCs-siCREG group was significantly increased  (P < 0.05). Western blot analysis and gelatinase spectrum analysis showed that MMP9 activity in the hVSMCs-siCREG group was significantly increased (P < 0.05) and at the same time, JNK protein was activated. Blocking study using JNK inhibitor confirmed that CREG promoted VSMCs migration by regulating JNK and MMP9. Results showed that CREG protein expression can inhibit the migration of VSMCs cultured in vitro by inhibiting the activation of JNK and MMP9.

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