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研究
发布时间：2019-03-12 09:48
【摘要】：目的:在建立蛋白质组分析方法的基础上,运用质谱技术并结合生物信息学方法,对前列腺素E2受体EP3的选择性激动剂ONO-AE-248诱导的中性粒细胞(polymorphonuclear neutrophil, PMN)非凋亡性程序化细胞死亡(non-apoptotic programmed cell death)和正常中性粒细胞自发性凋亡进行蛋白质组学研究,分析鉴定在这两种不同的生物学过程中的差异表达蛋白质,并动态观察这些差异蛋白质在PMN非凋亡性程序化细胞死亡过程中表达的动力学特征,以期为探讨“非凋亡性程序化死亡”这种新的细胞死亡方式的分子机制提供实验依据,为丰富细胞死亡的多样性奠定理论基础,同时也为重新认识和定位中性粒细胞在固有性免疫应答中的地位和作用提供新的思路。方法:采用梯度离心法分离、纯化正常人外周血中性粒细胞,调整细胞数为2×106/ml,以每孔1 ml接种于24孔培养板中。将分离的PMN随机分为实验组和对照组,在实验组中每孔均加入ONO-AE-248(终浓度为5×10-5 mol/ ml),对照组加入等量的培养基,将细胞置于37℃,CO2含量5%,湿度90%的CO2培养箱中分别孵育培养2 h、6 h、12h、24 h,构建PMN非凋亡性程序化死亡和自发性凋亡的细胞模型。在各时间点分别提取实验组和对照组PMN的全细胞蛋白质,Bradford法测定蛋白质含量,双向电泳(一向采用pH 4-7的固相pH梯度(IPG)等电聚焦,二向采用浓度为10%的十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白,银染后获得双向电泳图谱。凝胶成像系统采集电泳图谱,根据IPG胶条的线性梯度和标准分子量marker
[Abstract]:Objective: on the basis of establishing proteome analysis method, the neutrophil (polymorphonuclear neutrophil, induced by ONO-AE-248, a selective agonist of prostaglandin E 2 receptor EP3, was induced by mass spectrometry combined with bioinformatics method. PMN) non-apoptotic programmed cell death (non-apoptotic programmed cell death) and spontaneous apoptosis of normal neutrophils were studied by proteomics, and the differentially expressed proteins in these two different biological processes were analyzed and identified. The dynamic characteristics of these differentially expressed proteins in the process of non-apoptotic programmed cell death in PMN were observed dynamically in order to provide experimental basis for exploring the molecular mechanism of "non-apoptotic programmed death", a new way of cell death, in order to provide experimental basis for exploring the molecular mechanism of "non-apoptotic programmed death". It lays a theoretical foundation for enriching the diversity of cell death and provides a new way to re-understand and locate the role of neutrophils in the innate immune response. Methods: normal human peripheral blood neutrophils were isolated and purified by gradient centrifugation. The number of neutrophils was adjusted to 2 脳 10 ~ 6ml and inoculated into 24-well culture plate with 1 ml per well. The isolated PMN were randomly divided into the experimental group and the control group. ONO-AE-248 was added to each hole of the experimental group (the final concentration was 5 脳 10 mol/ ml), the control group was added with the same amount of medium), the cells were placed at 37 鈩，
本文编号：2438672
[Abstract]:Objective: on the basis of establishing proteome analysis method, the neutrophil (polymorphonuclear neutrophil, induced by ONO-AE-248, a selective agonist of prostaglandin E 2 receptor EP3, was induced by mass spectrometry combined with bioinformatics method. PMN) non-apoptotic programmed cell death (non-apoptotic programmed cell death) and spontaneous apoptosis of normal neutrophils were studied by proteomics, and the differentially expressed proteins in these two different biological processes were analyzed and identified. The dynamic characteristics of these differentially expressed proteins in the process of non-apoptotic programmed cell death in PMN were observed dynamically in order to provide experimental basis for exploring the molecular mechanism of "non-apoptotic programmed death", a new way of cell death, in order to provide experimental basis for exploring the molecular mechanism of "non-apoptotic programmed death". It lays a theoretical foundation for enriching the diversity of cell death and provides a new way to re-understand and locate the role of neutrophils in the innate immune response. Methods: normal human peripheral blood neutrophils were isolated and purified by gradient centrifugation. The number of neutrophils was adjusted to 2 脳 10 ~ 6ml and inoculated into 24-well culture plate with 1 ml per well. The isolated PMN were randomly divided into the experimental group and the control group. ONO-AE-248 was added to each hole of the experimental group (the final concentration was 5 脳 10 mol/ ml), the control group was added with the same amount of medium), the cells were placed at 37 鈩，
本文编号：2438672
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