缺血预处理对人肺缺血再灌注损伤的蛋白质组学研究

发布时间：2018-08-03 20:10

【摘要】： 缺血预处理是指预先给予一次或多次短暂的缺血,以增强组织对随后长时间缺血再灌注损伤的耐受性或适应性。缺血再灌注损伤常见于休克时微循环的疏通、冠状动脉痉挛的缓解、心脑血管栓塞再通、心肺手术后和心脏骤停后心肺脑复苏、断肢再植、器官移植血供恢复等,是引起随后器官功能障碍的主要原因之一。大量的研究表明缺血预处理能显著减轻随后的缺血再灌注损伤。肺缺血再灌注损伤多见于体外循环心内直视术、肺动脉袖状切除术、肺移植术及需要阻断一侧肺动脉才能进行的肺叶切除术中,对肺功能恢复不利,并可能引起术后并发症和影响预后。肺缺血预处理是防治肺缺血再灌注损伤最有效的措施,但其确切的保护机制尚未阐明。研究显示,给予缺血预处理的肺组织与未予缺血预处理的肺组织相比,在经过随后长时间的缺血再灌注后,表现出更好的缺血耐受性和再灌注耐受性,其中性白细胞浸润,肺水肿,局部出血和肺泡破裂均明显减少,其细胞结构,内环境平衡,微循环灌注,能量代谢,组织功能和遗传重组等方面具有更好的延续性和恢复能力。广大科学工作者从动物实验和临床实验对肺缺血预处理的肺保护机制进行了卓有成效的研究,发现腺苷、缓激肽、花生四烯酸代谢产物、阿片类、儿茶酚胺类、自由基、钙离子、一氧化氮和活性氧等与保护效应的启动相关;G蛋白、磷脂酶C、蛋白激酶A、酪氨酸蛋白激酶及丝裂原活化的蛋白激酶等与保护效应的介导相关,而保护效应的发挥则与ATP敏感钾通道、5'核苷酶与腺苷、转录因子、诱生型一氧化氮合成酶、环氧化酶2、醛糖还原酶、抗自由基酶类和热休克蛋白等相关。虽然已有的研究揭示了一些肺缺血预处理的保护机制,但目前仍然不能完全解释肺缺血预处理引起的保护效应并有效逆转缺血再灌注损伤。肺缺血预处理的临床研究开展较晚,但是缺血预处理作为大手术的辅助措施能够安全而巧妙地实施。蛋白质组学技术为研究缺血预处理的保护机制提供了新的工具。为此,本研究以临床中的手术患者为对象,采用蛋白质组学技术和方法研究缺血预处理抗人肺缺血再灌注损伤的分子机制。首先选择合适的手术患者分别归入假手术组(Sham group,S组)、缺血再灌注组(Ischemia-reperfusion group,I/R组)和缺血预处理组(Ischemic preconditioning group IP组)。I/R组经历单纯的缺血再灌注,IP组在缺血再灌注前予以缺血预处理,S组始终保持供血与通气,在术后收取标本。然后采用一步抽提法制备S组,I/R组和IP组肺组织的总蛋白质,应用双向凝胶电泳技术建立了重复性和分辨率均较好的S组,I/R组和IP组肺组织总蛋白质的双向凝胶电泳图谱。PDQuest软件对三组肺组织蛋白质的双向电泳图谱进行了分析比较,得出20个4倍以上差异表达的蛋白质点,采用MALDI-TOF-MS对差异表达的蛋白质进行质谱分析,获取MALDI-TOF-MS肽质量指纹图谱,数据库搜索鉴定了17个差异表达的蛋白质。最后,为了验证比较蛋白质组学研究结果,采用Western blot技术对部分差异表达蛋白质在IP组和I/R组肺组织中的表达水平进行了检测,其结果与比较蛋白质组学研究的结果一致。本研究在建立S组、I/R组和IP组肺组织总蛋白质双向电泳图谱的基础上,采用质谱分析结合数据库搜索共鉴定出17个明显差异表达的蛋白质,这些蛋白质按功能主要可以分为八类,即代谢相关酶类,抗氧化剂,贮铁蛋白,钙结合蛋白质,分子伴侣,细胞骨架蛋白质,细胞增殖相关蛋白,以及信号传导相关蛋白质。其中一些蛋白质与自发性抗缺血再灌注损伤有关,一些蛋白质如HSP27及Cu/ZnSOD则与缺血预处理抗缺血再灌注损伤的主动效应有关,还有一些蛋白质与缺血预处理抗缺血再灌注损伤的稳定效应有关。研究结果为揭示缺血预处理的肺保护机制,以及寻找逆转缺血再灌注损伤的方法提供了科学依据。
[Abstract]:Ischemic preconditioning refers to the preconditioning of one or more transient ischemia to enhance the tolerance or adaptability of the tissue to the subsequent long ischemic reperfusion injury. Ischemia reperfusion injury is common to the dredging of microcirculation during shock, remission of coronary spasm, cardio cerebral vascular embolization, cardiopulmonary surgery and cardiac arrest after cardiopulmonary resuscitation. A large number of studies have shown that ischemic preconditioning can significantly reduce subsequent ischemia-reperfusion injury. Lung ischemia reperfusion injury is often seen in cardiopulmonary bypass, pulmonary sleeve resection, lung transplantation, and the need to block one. During lobectomy of the lateral pulmonary artery, it is unfavorable to the recovery of lung function and may cause postoperative complications and prognosis. Pulmonary ischemic preconditioning is the most effective measure for the prevention and treatment of pulmonary ischemia reperfusion injury, but the exact mechanism of protection has not been clarified. The study showed that the lung tissue was given ischemic preconditioning with the preconditioning without ischemic preconditioning. Compared to the lung tissue, after a long period of ischemia and reperfusion, it showed better ischemic tolerance and reperfusion tolerance, including leukocyte infiltration, pulmonary edema, local hemorrhage and alveolar rupture, and its cellular structure, internal environment balance, microcirculation perfusion, energy metabolism, tissue function and genetic recombination. There are better continuity and resilience. Many scientists have conducted fruitful studies on lung protection mechanism of lung ischemic preconditioning from animal experiments and clinical trials, and found that adenosine, bradykinin, peanut four enoic acid metabolites, opioids, catecholamines, free radicals, calcium ions, nitric oxide and reactive oxygen species and other protective effects G proteins, phospholipase C, protein kinase A, tyrosine protein kinase and mitogen activated protein kinase are related to the protective effects, while the protective effects play with the ATP sensitive potassium channel, 5'nucleoside and adenosine, transcription factors, inducible nitrogen oxide synthase, cyclooxygenase 2, aldose reductase, and anti free radical enzymes. Although some studies have revealed protective mechanisms of lung ischemic preconditioning, there is still no complete explanation of the protective effects of lung ischemic preconditioning and the effective reversal of ischemia-reperfusion injury.
The clinical study of pulmonary ischemic preconditioning is carried out later, but ischemic preconditioning can be implemented safely and skillfully. Proteomics technology provides a new tool to study the protective mechanism of ischemic preconditioning. For this reason, this study uses proteomics techniques and methods in clinical patients. Objective to study the molecular mechanism of ischemic preconditioning against human lung ischemia-reperfusion injury.
First, the appropriate surgical patients were selected for the sham operation group (Sham group, group S), the ischemic reperfusion group (Ischemia-reperfusion group, I/R group) and the ischemic preconditioning group (Ischemic preconditioning group IP group).I/R group underwent simple ischemia reperfusion. The IP group was pretreated with ischemia before reperfusion. The total protein of lung tissue in group S, I/R group and IP group was prepared by one step extraction. The two way gel electrophoresis technique was used to establish the S group with good reproducibility and resolution. The bidirectional gel electrophoresis of the total protein of lung tissue in group I/R and IP group.PDQuest software for the three groups of lung tissue proteins. In the analysis and comparison of the swimming atlas, 20 protein points with 4 times of differential expression were obtained. MALDI-TOF-MS was used to analyze the proteins expressed differently, to obtain the mass fingerprint of MALDI-TOF-MS peptide, and to identify 17 differentially expressed proteins by database search. Finally, to verify the results of comparative proteomics, use We Stern blot technique was used to detect the expression level of partially differentially expressed proteins in the lung tissue of group IP and I/R group, and the results were in agreement with the results of comparative proteomics.
In this study, on the basis of the bi-directional electrophoresis of the total protein of lung tissue in group S, group I/R and IP, 17 proteins with distinct differentially expressed proteins were identified by mass spectrometry analysis and database search. These proteins can be divided into eight categories according to their function, namely, metabolic enzymes, antioxidants, ferric proteins, calcium binding proteins, and molecules. Chaperones, cytoskeleton proteins, cell proliferation related proteins, and signal transduction related proteins. Some proteins are associated with spontaneous anti ischemia-reperfusion injury, and some proteins such as HSP27 and Cu/ZnSOD are associated with the active effect of ischemic preconditioning against ischemia-reperfusion injury, and some proteins and ischemic preconditioning. The results of the study provide a scientific basis for revealing the mechanism of ischemic preconditioning and finding a way to reverse the injury of ischemia-reperfusion.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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