【作者】 刘骥；

【导师】 邓小明； 李金宝；

【作者基本信息】 第二军医大学 ， 麻醉学， 2009， 博士

【摘要】 【目的】脓毒症的早期诊断有助于采取更为恰当的治疗措施以改善脓毒症患者的预后,但目前仍缺乏有效的预测手段。本研究应用双向电泳和生物质谱鉴定的差异蛋白质组学方法寻找脓毒症患者血小板中差异表达蛋白,并对这些差异表达蛋白分子进行进一步的研究和筛选,以期寻找到脓毒症相关的血小板特异性蛋白标志物,同时进一步明确脓毒症血小板蛋白质组病理生理变化的分子机制,以便早期采取积极措施以改善脓毒症预后。【方法】新鲜血液采集自十日内未用过任何药物的正常健康受试者以及脓毒症确诊24 h的脓毒症患者,每份血样都单独处理,加入体积比10%的枸橼酸钠抗凝在室温下200g离心20 min获得富血小板血浆(PRP)。取PRP的上三分之一入新试管加入最终浓度为2.5mM的前列环素,1000g离心10min。去除血浆后,用Tyrodes-HEPES液洗涤吹悬血小板至浓度2×108/ml,室温下孵育30min,15000g离心裂解血小板后加入20 l蛋白酶抑制剂,-80°C冻存。利用固相pH梯度及SDS-PAGE二维凝胶电泳分离PLT总蛋白,考马斯亮蓝染色、Imagescanner扫描、PDQuest软件图像分析找出差异表达蛋白质点,应用基质辅助激光解吸电离飞行时间质谱分析(MALDI-TOF-TOF)及Mascot查询软件、SwissPort数据库鉴定差异表达蛋白。【结果】1.优化获得了重复性和分辨率较好的正常健康受试者及脓毒症患者血小板蛋白质组双向凝胶电泳(2-DE)图谱。2.建立了正常健康受试者与脓毒症患者血小板2-DE差异表达图谱。3.采用质谱法鉴定了2-DE图谱上的差异蛋白质点,通过MALDI-TOF-MS检测和蛋白质数据库搜寻,成功鉴定了6个蛋白质点,分别为来自细胞骨架蛋白家族的Actin, cytoplasmic 1;信号转导通路蛋白家族的IL-1β、EF-hand calcium-bindingdomain-containing protein 7;膜受体蛋白家族的GPⅨ、GPⅡb/Ⅲa和翻译后加工蛋白Protein disulfide-isomerase A3。【结论】脓毒症后血小板蛋白质组与正常受试者相比差异显著。应用基于2-DE和质谱分析的蛋白质组学方法可全面了解血小板蛋白质组变化规律,采用差异蛋白质组学成功鉴定了脓毒症血小板蛋白质组中6个差异表达蛋白,这些蛋白包括细胞骨架蛋白家族、信号转导通路蛋白家族、膜受体蛋白家族、翻译后加工蛋白。提示联合应用2-DE和MS技术的蛋白质组学在在筛查脓毒症临床诊断标志物方面是一个强有力的工具,有望用于临床。更多还原

【Abstract】 ObjectiveEarly diagnostic of sepsis is an attractive strategy to decrease the mortality of septic patients, but presently there are no satisfactory approaches. This study employed the technology of 2-DE and MS to look for the differential proteins of platelets between the normal healthy volunteers and septic patients and then to explore some potential biomarkers related with sepsis.MethodsFresh blood was collected from normal healthy volunteers who were not on medication for the previous ten days and septic patients. Each blood sample was processed individually and was mixed with a sodium citrate stock solution to final concentration of 10% v/v of the anticoagulant. the blood was centrifuged for 20 min at 200 g at room temperature to obtain platelet rich plasma (PRP). The upper third of the PRP was recentrifuged in a fresh tube removing any contaminating leukocytes. Addition of prostacyclin (final concentration 2.5 mM) allowed pelleting of platelets at 1000g with minimal activation. Following removal of plasma, the platelets were washed in Tyrodes-HEPES. The platelet pellet was then resuspended in Tyrodes-HEPES, to a concentration of 2×108/mL or 1×109 /mL, and incubated at room temperature for 30 min. Platelets were spun down at 15000g in the absence of prostacyclin and, following addition of 20 l of a protease inhibitor cocktail, immediately frozen in liquid nitrogen prior to storage at–80℃for 2-DE, image analysis and MALDI-TOF-MS.Results1. Platelet 2-DE profiles of normal healthy volunteers and septic patients with high resolution and reproducibility were obtained.2. Differrential platelet 2-DE profiles between normal healthy volunteers and septic patients were established.3. Differential protein spots between normal healthy volunteers and septic patients platelet 2-DE profiles were identified by MALDI-TOF-MS and protein datebase from internet. There are 6 differential protein spots were identified. They are belong to membrane receptors, signalling molecules, cytoskeletal and protein processing respectively.ConclusionsAfter sepsis, the proteome of septic patinets’platelet changes markedly. The technology of proteomics based on 2-DE and MS offers a huge potential for the provision of comprehensive protein expression date for basal and activated plateltes. Employed this proteomic approach, we successfully identified 6 proteins in septic patients’platelet protome. They are GPⅨ, GPⅡb/Ⅲa belong to membrane receptors family, IL-1β, EF-hand calcium-binding domain-containing protein 7 belong to signalling molecules family, Actin, cytoplasmic 1 belong to cytoskeletal family and protein disulfide-isomerase A3 belong to protein processing family respectively. The proposed approach combined use of 2-DE and MS provides an effective and high-throughput aproach for screening clinical diagnostic makers of septic patients.更多还原