CAJ | 학술논문

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.
목적 비교선립체DNA(mtDNA)결실A549세포(Rho0세포)여기모본세포(Rho+세포)핵단백표체보,병탐토세포핵대선립체공능결함적응답반응.방법 이유응효전영(2-DE)화표면증강격광법해흡전리-비행시간(SELDI-TOF)단백심편측정Rho0세포화Rho+세포핵단백표체보,기질보조격광해흡전리-비행시간(MALDI-TOF)질보결합수거고검색감정차이표체적단백점,Western인적법측정핵린단백화P53표체,격광공취초현미경측정선립체막전위.결과 2-DE현시Rho0세포핵중11개단백점표체하조,21개단백점표체상조.기우NP20단백질심편적SELDI-TOF질보분석발현4개단백질봉재Rho0세포핵중명현하강.기중1개표체하조적단백점피감정위eIF-6,4개표체상조적단백점피감정위핵린단백,SFRS1,SFRS3화hnRNP G.Western인적실험결과현시,Rho0세포중핵린단백표체증가.P53화선립체막전위(MMP)측정결과현시,Rho0세포중P53표체고우Rho+세포,량충세포MMP기본일치.결론 mtDNA결실유도료세포핵단백질조개변.Rho0세포가이작위연구선립체여핵교호작용적모형.
OBJECTIVE To investigate the nuclear proteomes in mitochondrial DNA (mtDNA)-depleted A549 cells (Rho0 cells) and their parental cells (Rho+ cells),and to learn more about the nuclear responses to mitochondrial dysfunction.METHODS The nuclear proteomes of Rho and Rho + cells were characterized by two dimensional electrophoresis (2-DE) and SELDI-TOF ProteinChip technologies,the differentially expressed protein- spots were identified by MALDI-TOF mass spectrum (MS),the nucleophosmin and P53 expression were detected by Western blotting assay,and the mitochondrial memhrane potential (MMP) was measured by the laser scanning confocal microscope.RESULTS 2-DE results showed 11 protein-spots were down-regulated and 21 protein-spots were up-regulated in Rho0 cell nuclei.SELDI-TOF MS analysis with NP20 ProteinChips revealed 4 protein-peaks decreased in Rho0 cell nuclei.One down-regulated protein-spot was identified as elF-6,and 4 up-regulated proteinspots were identified as nucleophosmin,SFRS1,SFRS3 and hnRNP G,respectively.The increased expression of nucleophosmin in Rho0 cells was verified by Western blotting.Based on the clues from proteomic analysis,P53 expression in Rho0 cells was higher than in Rho + cells,and MMP was consistent in Rho + and Rho0 cells.CONCLUSION mtDNA-depletion induces nuclear proteome alteration.Rho0 cells can be used as a model to study the crosstalk between mitochondrion and nucleus.