Objective To investigate the effects of different isolation time spacing on proteome profiles of peripheral blood mononuclear cells (PBMC), and find the most suitable PBMC isolation time spacing in clinical and fundamental research. Methods PBMC were isolated by Ficoll-Hypaque density gradient centrifugation from blood samples 0, 2, 4, 6, 8, 12h after being collected, and were observed under microscope. Proteins extracted from the PBMC were separated by SDS-PAGE, and the differently expressed proteins were analyzed and identified by liquid chromatography coupled with ion trap mass spectrometry. The functions and locations of these differential proteins were analyzed by consulting the GO database. Results The PBMC were not intact when the isolation time spacing was 12 h. More differential proteins were observed when the isolation time spacing was increased. In all the differently expressed proteins, most were transportation and signal transduction proteins and were located in nucleus and cell membrane when the isolation time spacing were 2,4, and 6 h, which were significantly different from those at 8 h. Conclusion The proteomes were successfully used in evaluating the dynamic variation of PBMC proteins when different isolation time spacing were performed, the most suitable and feasible PBMC isolation time spacing were 6 h.%目的:分析不同分血时间对外周血单个核细胞(PBMC)蛋白质表达谱的影响,探讨临床和研究中合适可行的PBMC分血时间.方法:在采血后0、2、4、6、8、12 h采用Ficoll-Hypaque密度梯度离心法分离血液中的PBMC,显微镜观察细胞形态;选取分血时间为0、2、4、6、8 h的PBMC提取蛋白质进行蛋白质凝胶电泳,选取差异表达蛋白质条带,通过液相色谱串联离子井质谱鉴定差异表达的蛋白质; 结合GO数据库信息对差异表达蛋白质的功能和定位进行分析.结果:分血时间为12 h已观察不到完整的PBMC细胞形态;质谱鉴定表明随着分血时间的延长,差异蛋白质逐渐增多;在2、4、6 h鉴定出的功能和定位明确的差异蛋白质中,大部分蛋白质功能为转运和信号转导,定位在细胞核和细胞膜上;而8 h鉴定出的差异蛋白质的功能的定位发生了显著的变化.结论:蛋白质组学技术能有效地观察不同分血时间导致的PBMC蛋白质表达的动态变化,推荐合适且临床可行的PBMC分血时间为6 h以内.
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