發(fā)布時(shí)間：2019-03-12 09:48

【摘要】：目的:在建立蛋白質(zhì)組分析方法的基礎(chǔ)上,運(yùn)用質(zhì)譜技術(shù)并結(jié)合生物信息學(xué)方法,對(duì)前列腺素E2受體EP3的選擇性激動(dòng)劑ONO-AE-248誘導(dǎo)的中性粒細(xì)胞(polymorphonuclear neutrophil, PMN)非凋亡性程序化細(xì)胞死亡(non-apoptotic programmed cell death)和正常中性粒細(xì)胞自發(fā)性凋亡進(jìn)行蛋白質(zhì)組學(xué)研究,分析鑒定在這兩種不同的生物學(xué)過程中的差異表達(dá)蛋白質(zhì),并動(dòng)態(tài)觀察這些差異蛋白質(zhì)在PMN非凋亡性程序化細(xì)胞死亡過程中表達(dá)的動(dòng)力學(xué)特征,以期為探討“非凋亡性程序化死亡”這種新的細(xì)胞死亡方式的分子機(jī)制提供實(shí)驗(yàn)依據(jù),為豐富細(xì)胞死亡的多樣性奠定理論基礎(chǔ),同時(shí)也為重新認(rèn)識(shí)和定位中性粒細(xì)胞在固有性免疫應(yīng)答中的地位和作用提供新的思路。方法:采用梯度離心法分離、純化正常人外周血中性粒細(xì)胞,調(diào)整細(xì)胞數(shù)為2×106/ml,以每孔1 ml接種于24孔培養(yǎng)板中。將分離的PMN隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組,在實(shí)驗(yàn)組中每孔均加入ONO-AE-248(終濃度為5×10-5 mol/ ml),對(duì)照組加入等量的培養(yǎng)基,將細(xì)胞置于37℃,CO2含量5%,濕度90%的CO2培養(yǎng)箱中分別孵育培養(yǎng)2 h、6 h、12h、24 h,構(gòu)建PMN非凋亡性程序化死亡和自發(fā)性凋亡的細(xì)胞模型。在各時(shí)間點(diǎn)分別提取實(shí)驗(yàn)組和對(duì)照組PMN的全細(xì)胞蛋白質(zhì),Bradford法測(cè)定蛋白質(zhì)含量,雙向電泳(一向采用pH 4-7的固相pH梯度(IPG)等電聚焦,二向采用濃度為10%的十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分離蛋白,銀染后獲得雙向電泳圖譜。凝膠成像系統(tǒng)采集電泳圖譜,根據(jù)IPG膠條的線性梯度和標(biāo)準(zhǔn)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，

本文編號(hào)：2438672