中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (7): 1322-1326.doi: 10.3969/j.issn.1673-8225.2010.07.043

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

去除蛋白激酶A磷酸化位点的线粒体融合素2基因与血管平滑肌细胞增殖

周  炜1，曹文静2，陈莉莉3，郭小梅1，陈光慧○4   

  1. 华中科技大学同济医学院附属同济医院，1心内科，2检验科，3老年科，湖北省武汉市430030；4美国国家卫生研究所心血管研究室(National Institutes of Health，NIH)，9000 Rockville Pike Bethesda, Maryland 20892，USA
  • 出版日期:2010-02-12 发布日期:2010-02-12
  • 通讯作者: 郭小梅，博士，教授，华中科技大学同济医学院附属同济医院心内科，湖北省武汉市 430030
  • 作者简介:周 炜☆，男，1979年生，湖北省武汉市人，汉族，2002年华中科技大学同济医学院毕业，博士，主要从事冠心病的介入治疗和分子心脏病学研究。
  • 基金资助:

    国家自然科学基金资助项目(30672206；30600233)**

Effect of mitofusin 2 gene with protein kinase A phosphorylation site deletion on the proliferation of vascular smooth muscle cells

Zhou Wei1, Cao Wen-jin2, Chen Li-li3, Guo Xiao-mei1, Chen Guang-hui○4   

  1. 1 Department of Cardiology, 2 Department of Clinical Laboratory, 3 Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan   430030, Hubei Province, China; 4 National Institute of Health, 9000 Rockville Pike Bethesda, Maryland 20892, USA
  • Online:2010-02-12 Published:2010-02-12
  • Contact: Guo Xiao-mei, Doctor, Professor, Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China xmguo@tjh.tjmu.edu.cn
  • About author:Zhou Wei☆, Doctor, Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China betaloc25mg@yahoo.com.cn
  • Supported by:

     the National Natural Science Foundation of China, No.30672206*, No.30600233*

PDF

1188

被引次数

4

摘要：

背景: 线粒体融合素2基因作用于血管平滑肌细胞Ras蛋白，通过胞外信号调节蛋白激酶1/2通路抑制细胞增殖。线粒体融合素2基因氨基酸序列第442位丝氨酸为蛋白激酶A磷酸化位点，与其磷酸化状态密切相关，可能参与其功能调控。
目的：观察大鼠线粒体融合素2基因在去除蛋白激酶A磷酸化位点后对大鼠血管平滑肌细胞增殖的影响及其相关信号通路。
方法：利用已构建的携带绿色荧光蛋白基因、线粒体融合素2基因和去除蛋白激酶A磷酸化位点的线粒体融合素2基因的3种重组腺病毒，感染大鼠主动脉血管平滑肌细胞，将其传代培养3~10代后以抽签法随机分为4组：①不加干预的对照组。②感染携带绿色荧光蛋白的对照组(Adv-GFP组)。③感染携带线粒体融合素2基因的实验组(Adv-Mfn2组)。④感染携带去除蛋白激酶A磷酸化位点的线粒体融合素2基因的实验组(Adv- Mfn2-PKA(△)组)。激光共聚焦显微镜观察完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在细胞中的定位。Western blot检测p-ERK1/2表达水平及完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中的表达。MTT法绘制细胞生长曲线。
结果与结论：完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中均表达蛋白特异性条带。两种基因表达产物都主要分布于线粒体外膜。与对照组和Adv-GFP组相比，Adv-Mfn2组吸光度值在第3，4，5，6天都显著降低(P < 0.01)，Adv-Mfn2-PKA(△)组吸光度值无明显变化。与对照组和Adv-GFP组相比，Adv-Mfn2组p-ERK1/2表达水平显著降低(P < 0.01)，Adv-Mfn2-PKA(△)组无明显变化。提示去除蛋白激酶A磷酸化位点的线粒体融合素2基因定位于线粒体外膜，对血管平滑肌细胞的增殖无拮抗作用，对胞外信号调节蛋白激酶1/2通路无抑制作用。

关键词: 线粒体融合素2基因, 血管平滑肌细胞, 增殖, 磷酸化, 组织工程

Abstract:

BACKGROUND: The mitofusin 2 (Mfn2) may affects vascular smooth muscle cell Ras protein and suppress cellular proliferation through inhibition of extracellular signal-regulated protein kinase signaling pathway, which plays an important role in pathogenesis of vascular disorders such as hypertension, atherosclerosis and post-angioplasty restenosis. Mfn-2 gene amino acid sequence of the first 442 serine serves as protein kinase A (PKA) phosphorylation site, which is closely related to its phosphorylation status and may be involved in its functional regulation.
OBJECTIVE: To investigate the effect of Mfn2 gene with PKA phosphorylation site deletion [Mfn2-PKA (△)] on inhibiting the proliferation of vascular smooth muscle cells and related signaling pathway.
METHODS: Vascular smooth muscle cells of rats infected by recombinational adenovirus carrying green fluorescent protein, Mfn2 gene and Mfn2-PKA (△), were subcultured for 3-10 passages and randomly divided into 4 groups: ① Control group without intervention. ② Control group infected with adenovirus carrying green fluorescent protein. ③ Experiment group infected with adenovirus carrying Mfn-2 gene. ④ Experiment group infected with adenovirus carrying Mfn2-PKA (△). Laser confocal microscopy was used to observe the locations of Mfn2 gene with and without PKA in cells. The expressions of extracellular signal-regulated protein kinase, Mfn2 gene and Mfn2-PKA (△) were determined by Western blot analysis. The growth curve of the vascular smooth muscle cells was explored by MTT.
RESULTS AND CONCLUSION: The Mfn-2 and Mfn2-PKA (△) both expressed protein-specific bands in vascular smooth muscle cells. Two kinds of gene expression products were mainly located at the out membrane of mitochondria. Compared with the control group and adenovirus carrying green fluorescent protein group, the absorbance values at 3, 4, 5, 6 days were significantly reduced in adenovirus carrying Mfn2 group (P < 0.01), and no obvious changes were observed in adenovirus carrying Mfn2-PKA (△) group. Compared with the control group and adenovirus carrying green fluorescent protein group, the extracellular signal-regulated protein kinase expression was significantly reduced in adenovirus carrying Mfn2 group (P < 0.01), and no obvious changes were observed in adenovirus carrying Mfn2-PKA (△) group. Mfn2-PKA (△) located at the out membrane of mitochondria, has no effect on suppressing the proliferation of vascular smooth muscle cells, and no inhibition effect on extracellular signal-regulated protein kinase signaling pathway.

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