ONO-AE-248诱发中性粒细胞非凋亡性程序化死亡的蛋白质组学分析方法的建立

目的:建立双向电泳技术和质谱技术在探索ONO-AE-248诱发的中性粒细胞(neutrophil,PMN)非凋亡性程序化死亡的分子机理研究中的应用,为深入认识“非凋亡性程序化死亡“这种新的死亡方式提供新的有效的研究途径.方法:将采用梯度离心法分离的,人正常PMN,分为正常对照组和实验组,实验组用ONO-AE-248刺激12小时建立非凋亡性坏死模型,提取对照组和实验组全蛋白,双向电泳分离蛋白质,采用PDQuest 2-DE软件分析找出两组间差异表达的蛋白质斑点,用基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF-MS)进行鉴定.结果:双向电泳图谱显示:ONO-AE-248诱导的人PMN非凋亡性程序化细胞死亡与PMN的自发性凋亡的蛋白质组存在差异显著,质谱鉴定初步得到其中12种差异表达的蛋白质.结论:双向电泳和质谱鉴定技术可以有效的分离和分析ONO-AE-248诱发的PMN非凋亡性坏死的蛋白质组,为进一步探索“非凋亡性程序化死亡“方式提供了新的方法和途径,为丰富细胞死亡方式带来了新的前景.

Develop the proteomic methords in research on ONE-AE-248-induced non-apoptotic programmed cell Death of neutrophils

Objective:To develop the proteomic methords in exploring the different mechanisms between ONO-AE-248-induced non-apoptosis programmed cell death of polymorphonuclear neutrophils (PMN) and normal apoptosis of PMN. Methods:PMN isolated from healthy volunteers by Ficoll were incubated in the presence or absence of ONO-AE-248 for 12 h. PMN proteins were extracted at the indicated time.The proteins were separated by 2-DE and then stained by silver staining.After being acquired by scanning Two-dimensional electrophoresis maps were analyzed by PDQuest2-DE software. 20 differentially expressed proteins in 12h-experimental group and 12h-control group were found. Results: (1)The proteomes between ONO-AE-248-induced non-apoptotic programmed cell death and normal apoptosis of PMN were obviously different. (2)By MALDI-TOF-MS and bioinformatics analysis, 12 differentially expressed proteins were identified. Conclusion: The methords of 2-DE and MALDI-TOF-MS utilized in this study have effectivly seperated and identified the proteins in ONO-AE-248-induced non-apoptotic programmed cell death, and provieds a valuble way to study the mechanism of ONO-AE-248-induced non-apoptotic programmed cell death.