Bak在细胞凋亡期间对线粒体形态学的影响

Effect of Bak on mitochondrial morphplogy in apoptosis

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10mmol.L-1叠氮钠或1μmol.L-1十字孢碱(STS)或20μmol.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。

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 fibrohlasts （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 ceils 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.