天津医药

• 实验研究 •    

Bax在14-3-3γ对抗脂多糖诱导的心肌细胞损伤中所起的作用

刘丹1,尹东2,孙惦1,许旻1,何明1   

  1. 1. 南昌大学医学院药理教研室
    2. 江西省分子医学重点实验室
  • 收稿日期:2012-02-28 修回日期:2012-07-11 出版日期:2012-12-15 发布日期:2012-12-15
  • 通讯作者: 刘丹

The Role of Bax in the 14-3-3γ Protection against Cardiomyocytes Damage Induced by LPS

  • Received:2012-02-28 Revised:2012-07-11 Published:2012-12-15 Online:2012-12-15
  • Contact: Dan Liu

摘要： 摘要 目的：研究导入外源性14-3-3γ基因是否能对抗脂多糖（LPS）所致心肌损伤，以及阐明Bcl-2家族中的Bax基因在此过程中所起的作用。方法: 培养原代SD乳鼠心肌细胞，构建重组质粒pFLAG-14-3-3γ转染至心肌细胞，行LPS损伤处理。实验结束后，Western blotting检测细胞中14-3-3γ蛋白表达情况，四唑盐比色试验检测细胞存活率，全自动生化分析仪检测LDH、CPK值，Western Blotting检测细胞浆及线粒体的Bax蛋白水平。结果：Western Blotting证实pFLAG-14-3-3γ转染至心肌细胞使细胞中14-3-3γ蛋白表达明显升高。LPS致心肌细胞损伤，与正常组相比，LPS处理使细胞存活率明显下降（P<0.01），LDH、CPK值明显升高（P<0.01），促使Bax蛋白从胞浆向线粒体移位，转染重组质粒pFLAG-14-3-3γ使14-3-3γ在心肌细胞内高表达后，则可明显逆转LPS所致的损伤，与未转染pFLAG-14-3-3γ组相比，转染组心肌细胞存活率明显升高（P<0.01），LDH、CPK值明显下降（P<0.01），并抑制Bax蛋白向线粒体的移位。结论：14-3-3γ能对抗LPS所致的心肌细胞损伤，其机制与抑制Bax从胞浆向线粒体移位有关。

关键词: 脂多糖, 心肌损伤, 基因重组, 14-3-3γ, 转染, Bax

Abstract: Abstract Objective: To investigate the exogenous gene 14-3-3γ against cardiomyocyte damage induced by LPS, and explore the role of Bax of Bcl-2 family involving in the process. Methods: Neonatal SD rat cardiomyocytes were cultivated and constructed recombinant plasmid of pFLAG-14-3-3γ were transfected into the cells, which were treated with LPS subsequently. After treatment, 14-3-3γ levels were detected by western blotting, cells viability was analyzed by MTT, the activity of LDH and CPK were determined by auto-biochemistry analysator, the levels of Bax protein in cytoplasm and mitochondria were detected by western blotting. Results: 14-3-3γ protein significantly increased in the cardiomyocytes transfected by pFLAG-14-3-3γ. Compared with control group, cardiomyocytes treated with LPS had significantly reduced cells viability(P<0.01), increased LDH and CPK activities(P<0.01), and promoted Bax translocation from cytoplasm to mitochondria. Elevated l4-3-3γ protein resulted by transfection of pFLAG-14-3-3γ reversed cardiomyocytes damage induced by LPS. Compared with untransfected group, cardiomyocytes of transfected group had significantly increased cells viability(P<0.01), decreased LDH and CPK activities(P<0.01), and inhibited Bax translocation from cytoplasm to mitochondria. Conclusion: 14-3-3γ protects cardiomyocytes against LPS damage by inhibiting Bax translocation from cytoplasm to mitochondria.

Key words: LPS, myocardial damage, gene recombination, 14-3-3γ, transfection, Bax