以iTRAQ技术分析金雀异黄酮作用大鼠肝星状细胞差异表达的蛋白

Analysis of genistein-induced differentially expressed proteins in rat hepatic stellate cells by iTRAQ

目的:分析金雀异黄酮作用大鼠肝星状细胞(HSC-T6)后差异表达的蛋白质,以探讨金雀异黄酮抗肝纤维化的作用机制。方法:以金雀异黄酮作用HSC-T6细胞后,提取细胞总蛋白,采用同位素标记的iTRAQ技术结合高效液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)的定量蛋白质组学方法,分析和鉴定差异表达的蛋白质,运用生物信息学分析差异表达的蛋白质功能。结果:质谱共分析鉴定到685种蛋白质。3次实验鉴定到的差异蛋白质数量分别为79,88,75个,在3次重复实验均鉴定到的差异蛋白为44个,其中上调蛋白19个,下调蛋白25个。对这些差异蛋白进行功能分析,发现参与翻译后修饰、转录、重组和信号传导等过程。结论:iTRAQ联合LC-ESI-MS/MS技术能高通量筛选金雀异黄酮抗纤维化作用的蛋白质。金雀异黄酮的抗纤维化作用,可能是通过对蛋白质调控表达来实现。

Objective： To detect differences in protein expression after genistein treatment in rat hepatic stellate HSC-T6 cells,and to explore antifibrotic mechanism of genistein in the protein levels. Methods： HSC-T6 cells were used as the target cells. Proteins were extracted from the cells after treated by genistein. After equal amount of protein digested by trypsin,the iTRAQ-labeled proteins and peptides were separated by the cation column（SCX）,and differentially expressed proteins were detected and identified by LC-ESI-MS /MS. The functions of proteins were analyzed by bioinformatics. Results： Totally 685 proteins were identified by MS. The 79,88 and 75 differentially expressed proteins were identified in 3 separate experiments,respectively. Among these proteins,44 were always identified in the three experiments,of which 19 were up-regulated and 25 were down-regulated. The differentially expressed proteins correlated with post-translational modification,transcription,recombination and signal transduction. Conclusion： Genistein can regulate protein expression in HSC-T6 cells. Its antifibrotic effects may be due to regulation of protein expression.