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目的比较线粒体DNA(mtDNA)缺失A549细胞(Rho0细胞)与其母本细胞(Rho+细胞)核蛋白表达谱,并探讨细胞核对线粒体功能缺陷的应答反应。方法二维凝胶电泳(2-DE)和表面增强激光法解吸电离-飞行时间(SELDI-TOF)蛋白芯片测定Rho0细胞和Rho+细胞核蛋白表达谱,基质辅助激光解吸电离-飞行时间(MALDI-TOF)质谱结合数据库检索鉴定差异表达的蛋白点,Western印迹法测定核磷蛋白和P53表达,激光共聚焦显微镜测定线粒体膜电位。结果 2-DE显示Rho0细胞核中11个蛋白点表达下调,21个蛋白点表达上调。基于NP20蛋白质芯片的SELDI-TOF质谱分析发现4个蛋白质峰在Rho0细胞核中明显下降。其中1个表达下调的蛋白点被鉴定为eIF-6,4个表达上调的蛋白点被鉴定为核磷蛋白,SFRS1,SFRS3和hnRNP G。Western印迹实验结果显示,Rho0细胞中核磷蛋白表达增加。P53和线粒体膜电位(MMP)测定结果显示,Rho0细胞中P53表达高于Rho+细胞,两种细胞MMP基本一致。结论 mtDNA缺失诱导了细胞核蛋白质组改变。Rho0细胞可以作为研究线粒体与核交互作用的模型。
Objective To compare the nuclear protein expression profiles of mitochondrial DNA damage (MhDNA) and A549 cells (Rho0 cells) with its maternal cells (Rho + cells) and to investigate the response of nuclei to mitochondrial dysfunction. Methods Two-dimensional gel electrophoresis (2-DE) and surface enhanced laser desorption ionization-time of flight (SELDI-TOF) protein chip were used to determine the Rho0 cells and Rho + nuclear protein expression profiles, matrix-assisted laser desorption ionization-time of flight ) Mass spectrometry combined with database search to identify differentially expressed protein spots, nucleoside phosphoprotein and P53 expression were determined by Western blotting, and mitochondrial membrane potential was measured by confocal microscopy. Results 2-DE showed 11 protein spots in Rho0 nucleus were down-regulated and 21 spots were up-regulated. SELDI-TOF mass spectrometry analysis based on the NP20 protein chip revealed that four protein peaks were significantly decreased in the RhoO nuclei. One of the down-regulated protein spots was identified as eIF-6, and four of the up-regulated protein spots were identified as nucleophosmin, SFRS1, SFRS3 and hnRNPG. Western blotting results showed that Rho0 cells nuclear ribosomal protein expression increased. The results of P53 and mitochondrial membrane potential (MMP) showed that the expression of P53 in Rho0 cells was higher than that in Rho + cells, and the MMPs in the two cells were basically the same. Conclusion The deletion of mtDNA induced the alteration of nuclear proteome. Rho0 cells can be used as a model to study the interaction between mitochondria and nucleus.