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• 基础研究 • 上一篇    下一篇

Bak在细胞凋亡期间对线粒体形态学的影响

冯乐平1,乔 伟1, Craig Brooks2, Zeng Dong2   

  1. 1.桂林医学院生物技术学院, 广西 桂林 541004;2.佐致亚医学院细胞生物与解剖学系,佐治亚 奥古斯塔  30912
  • 收稿日期:2007-11-30 修回日期:1900-01-01 出版日期:2008-09-28 发布日期:2008-09-28
  • 通讯作者: 乔 伟

Effect of Bak on mitochondrial morphplogy in apoptosis

FENG Le-ping1,QIAO Wei1,CRAIG Brooks2,ZENG Dong2   

  1. 1.School of Biotechnology,Guilin Medical College,Guilin 541004,China;2.Department of Cellular Biology and Anatomy,Medical College of Geogia,Augusta GA 30912,USA
  • Received:2007-11-30 Revised:1900-01-01 Online:2008-09-28 Published:2008-09-28
  • Contact: QIAO Wei

摘要: 目的:探讨Bak基因在细胞凋亡线粒体信号传导中的作用以及与Bax基因之间的相互关系。方法:应用Bax/Bak双重基因敲除的小鼠胚胎成纤维细胞(MEF)和Hela细胞进行基因重构,细胞按不同基因型(野生型、Bax敲除和Bak敲除、双重基因敲除)分组,并通过共转染方式导入目的基因。细胞凋亡的处理条件:细胞在无葡萄糖缓冲液的培养基中,在含10 mmol•L-1叠氮钠或1 μmol•L-1十字孢碱(STS)或20 μmol•L-1顺铂条件下培养,并诱导细胞凋亡。应用共聚焦显微镜和免疫荧光及免疫印记技术分析细胞凋亡、线粒体碎裂和细胞色素C释放情况,综合评价Bak基因在细胞凋亡和线粒体碎裂中的作用。结果:Hela细胞内线粒体碎裂和细胞色素C释放百分率分别与细胞凋亡有关联(P<0.01);MEF细胞在Bak基因缺失(Bak-/-)时,应用化学诱 导剂导致的细胞凋亡率(17.9%)明显低于野生型MEF细胞(71.3%)(P<0.01);且Bax和Bak基因两者协同作用可以增强细胞凋亡发生率;当pEGFP-Bak重新导入Bax-/-/Bak-/-MEF细胞,线粒体破碎百分率从43.7%增加到76.4%,pEGFP-Bak组细胞凋亡率明显高于pEGFP-Bax组(P<0.05);应用不同的化学诱导剂均可以得到同样的结果;且Bcl-2不能够明显阻止Bak诱导的细胞凋亡。结论:Bak基因明确诱导线粒体碎裂和细胞色素C释放,导致细胞凋亡,其诱导细胞凋亡的效果明显大于Bax基因,并且其作用独立于Bax基因功能之外。

关键词: 细胞色素C , 线粒体, Bax基因, Bak基因

Abstract: Abstract:Objective To study the function of Bak in mitochondrial signaling pathway and interaction between Bax and Bak during apoptosis.Methods Bax/Bak double knock out (Bax-/- and Bak-/-)MEF cells from mouse embryo fibroblasts (MEF) and Hela cells were divided into four groups according the cell different genotypes(wild type,Bax-/-,Bak-/- and double knock out) and treated with different chemical reagents after co-transfection with Bax,Bak,Mito-Red and empty pEGFP vector.Apoptosis,mitochondrial morphology and cytochrome C release were detected with confocal microscope,immunofluoresence and Western blotting techniques.Results There were correlations between the percentage of Hela cell apoptosis and mitochondrial fission(%) as well as cytochrome C(%) (P<0.01).When Bak gene deletion,the apoptotic rate of MEF cells(17.9%) induced by chemical reagent was lower than that of wild type MEF cells(71.3%)(P<0.01); When gene double knock out MEF cells were transfected with GFP-Bak,mitochondrial fission incresed from 43.7% to 76.4%,the apopotic rate in pEGFP-Bak group was significantly higher than that in GFP-Bax group(P<0.05) and the same result was gotten when treated with different chemical reagents. Bcl-2 can not inhibit the apoptosis induced with Bak.Conclusion Bak can induce cell mitochondria fission and cytochrome C release and result in apoptosis.Bak can induce more cell apoptosis than Bax and the effect stands alone besides Bax action.

Key words: cytochrome C, mitochondria, Bax gene, Bak gene

中图分类号: 

  • Q255