【作者】 唐建；

【导师】 王建军；

【作者基本信息】 华中科技大学 ， 胸外科， 2011， 博士

【摘要】 背景：肺移植(lung transplantation, LT)是当前治疗终末期肺部疾病的唯一有效方法,自1983年第一例成功的临床异体肺移植完成,二十多年来,肺移植技术越来越多的运用于临床并取得了长足的发展。现全球每年肺移植数量超过2000例,截止至2010年,全球158个中心行各类肺移植术共32652例,平均中位生存期5.3年,五年生存率达到了52%,十年生存率达29%。肺移植的生存率随着各种肺保护技术,抗排斥反应技术,及综合学科技术的进步逐渐提高[1]。然而,围手术期死亡仍然是造成肺移植失败的一个主要因素,据统计,肺移植围手术期死亡的主要原因是肺缺血再灌注损伤(Ischemia/Reperfusion Injury, I/R Injury)引起的原发性移植物功能障碍(Primary Graft Dysfunction, PGD) [2]。尽管手术技术、肺保护技术、重症监护技术不断提高,肺缺血再灌注损伤诱发的原发性移植物功能障碍仍然是肺移植早期死亡的主要病因[3]。发生在移植术后72小时内的非特异性肺泡及间质损伤、肺水肿、低氧血症是肺缺血再灌注损伤的主要表现[4]。过去的20年,缺血再灌注损伤的机制越来越清楚地被阐明,新型肺保护液及肺保护技术的运用使得原发性移植物功能障碍的发生率从90年代初的30%降低至目前的12%左右[2]。然而,肺组织相对于其他实质性脏器有其自有的生理特性,肺组织对缺血及其他外源性环境刺激更为敏感,供肺的获取与保护较其他实质性脏器更为严格。真正适合作为供肺的来源的器官数量也远远少于诸如肝,肾,心等实质性脏器[5]。据统计,约3/4的终末期肺部疾病患者在等待合适的供体的过程中就已死亡[6],开发新型的具有保护甚至恢复作用的肺保存药物可以减轻移植术后缺血再灌注损伤的发生率,并扩大供肺的获取来源,具有十分重要的临床意义。T淋巴细胞表面分化抗原CD26/DPPⅣ是一个相对分子质量为110kDa的多功能Ⅱ型跨膜糖蛋白,其具有二酰二肽酶活性,并在多种细胞,如上皮细胞、T淋巴细胞和内皮细胞等表面表达,其在肺组织中也发现了较高的表达[7]。之前我们的研究发现,通过抑制供肺二肽酰肽酶Ⅳ(DPPⅣ酶)的活性能够明显地减轻大鼠移植肺的I/R损伤,并在短期内(2 h)达到改善移植肺功能(pulmonary function)的作用[8-9]。然而抑制供肺DPPⅣ酶活性对术后较长时间段肺功能的影响以及其减轻移植肺I/R损伤的具体机制尚不清楚,推测可能与CD26/DPPⅣ对炎症反应的调节作用及其底物对肺功能的保护作用有关[10]。本实验旨在探讨抑制供肺DPPⅣ酶活性对大鼠肺移植术后较长时间段肺功能的影响,并运用差异蛋白质组学(proteomics)技术找出供肺DPPIV酶抑制后肺组织的差异蛋白表达,探究DPPIV酶活性减轻肺移植术后缺血再灌注损伤的机制。目的：建立大鼠原位异体肺移植模型(rat lung transplantation model),研究供肺DPPⅣ酶活性抑制对大鼠肺移植术后较长时间段内(7d)肺功能的影响,并运用差异蛋白质组学技术找出供肺DPPⅣ酶抑制后(1d)肺组织的差异蛋白表达,探究DPPⅣ酶活性抑制减轻肺移植术后缺血再灌注损伤的机制。方法：以SD大鼠作为试验动物,运用改良的三套管法建立大鼠原位异体左肺移植模型。运用DPPⅣ酶特异性抑制剂AB192灌注并保存供肺18h后行移植术,观察其对肺移植术后缺血再灌注损伤及长期肺功能的影响。将运用AB192灌注保存的进行肺组织与对照组供肺行蛋白组学研究,即利用双向凝胶电泳(two-dimensional polyacrylamide electrophoresis,2D-PAGE)分离两组总蛋白,通过图像分析寻找表达差异的蛋白点,对其进行MALDI-TOF质谱分析。对经质谱鉴定的蛋白质ATⅢ应用酶联免疫法对其含量进行鉴定,研究并验证其对肺缺血再灌注损伤的保护作用。结果：建立了稳定大鼠原位肺移植模型。对照组大鼠至术后第7天全部死亡,实验组(DPPⅣ酶特异性抑制组)的大鼠均存活至术后第7天。与对照组比较,各实验组的PIP值降低(P<0.05),PO2值升高(P<0.05),W/D值降低(P<0.05),MPO活性及MDA含量降低(P<0.05),并且随着时间的推移,实验组的上述指标不断改善,至术后第7天,各项检测值接近正常。运用差异蛋白质组学技术找出供肺DPPⅣ酶抑制后肺组织的差异蛋白表达,比较术后第1d两组供肺组织2D-PAGE图谱,得到差异蛋白点87个。对其中15个差异蛋白点进行质谱肽质量指纹图分析,鉴定出多个差异蛋白。其中9个差异蛋白均在实验组中过表达,而在对照组组织中低表达或不表达,6个在对照组中过表达。发现ATⅢ对肺组织缺血再灌注损伤有保护作用。结论：特异性的DPPⅣ酶抑制能够明显的减轻大鼠肺移植术后移植肺的缺血再灌注损伤,并有促进肺功能恢复作用。通过2-DE蛋白电泳成功的建立了大鼠肺组织2-DE图谱,并发现了多个差异蛋白表达,鉴定了其中15个蛋白质。发现了多个对肺功能有保护作用的蛋白及多种细胞信号转导通路蛋白。其中ATⅢ可能是DPPⅣ酶可能存在的对肺功能有保护作用的底物。更多还原

【Abstract】 Background:lung transplantation is the unique effective treatment for terminal pulmonary diseases, since the first allo-graft lung transplatation was accomplished successfully in 1983, it was used more and more in clinic and developed sufficiently. Currently, more than 2000 cases are completed in the world every year. By 2010, 32652 cases are accomplished in 158 centers worldwide, and the median survival time is 5.3y, the survival rate of 5 and 10 year are 52% and 29%. The survival rate is raised with the development of lung protective technique, anti-rejection technique, and combination of vary departments[1].The peroperative death is still a significant factor causing the failure of lung transplantation, according to statistics, the main cause of death is primary graft dysfunction(PGD) induced by ischemia reperfusion(I/R) injury[2]. Although the skills of operation, techniques of lung protection and intensive care therapy are improving, PGD is still the main cause of death in the early stage of lung transplantation[3]. Nonspecific alveolus injury, pulmonary edema, and hypoxemia within 72hrs post-transplantation are the major manifest of ischemia reperfusion injury[4]. In the previous two decades, the mechanisms of IR injury were interpreted more and more perspicuously, the use of new lung protective fluid and technology reduced the rate of PGD from 30% in 1990s to 12% presently. However, the lung has its specific physiological nature via other parenchymatous organs[5], and is more sensitive to ischemia and other ectogenesis enviromental stimulus, the acquisition and protection of lung are more strict than others. According to statistics, about 75% patient with terminal pulmonary diseases died during the waiting for suitable donors[6]. With the purpose of the release of IR injury and enlargement of donor sources, it is important to exploit new medicines for the storage of lung which can protect even revive lung functions.T lymphocyte surface differentiated antigen CD26/DPPIV is a multifunctional typeⅡtransmembrane glycoprotein with relative molecular mass of 110kDa, which possess dipeptidase enzyme activity and express on the surfaces of varied cells such as epithelial cells, T lymphocytes, and endotheliocytes. Its over-expression is also found in lung tissues[7]. Our research showed that I/R injury in rat lung transplantation can be released obviously by inhibiting the activity of DDPⅣenzyme in the donor lung, and pulmonary fuction can be improved in short-term(2h) [8-9]. However, the effects of inhibiting the activity of DDPⅣenzyme for long-term lung function after transplantation and the concrete mechanism for relieving IRI of transplanted lung are not clear yet. It is suspected to be related to the regulation of inflammation by DDPIV enzyme and the protection of lung function by its substrate[10]. By this experiment, we intend to investigate the effects of donor’s DPPIV enzyme activity inhibition on long-term lung function post-operation, and investigate the expression of differential proteins in lung tissue after inhibiting donor’s DPPIV enzyme activity by the methods of differential proteomics, to clarify the mechanisms of relieving I/RI after lung transplantation with the activity of DDPIV enzyme inhibition.Objective:To setup rat lung transplantation model, study the effect of inhibition of donor’s inhibiting donor’s DPPIV enzyme activity on long time lung function post-transplantation, and to find the expression of differential proteins in lung tissue after inhibiting donor’s DPPⅣenzyme activity at 1d post-transplantation by the technique of differential proteomics, and probe the mechanism of relieving IR injury after lung transplantation by inhibiting DPPIV enzyme activity.Methods:SD rats for experimental animals, we use modified "tri-cannulations" model to set up rat in-situ allograft lung transplantation. Donor lung tissue was perfused with specific DPPⅣenzyme inhibitor AB192 and preserved for 18h before transplantation, I/R injury after transplatation and effects for long-term lung function were observed. Proteomics was studied betweem the lung tissues perfused and preserved with AB192 and the non-interference ones in control groups, then two set of total protein were separated by two-dimensional polyacrylamide electrophoresis and differentially expressed protein site were searched. Under using MALDI-TOF mass chromatographic analysis, the proteins were detected by mass spectrum. Finally, we validate the protein by euzymelinked immunosorbent assay, and the use the protein to investigate its effect on lung I/R injury.Result:Stable pattern of in situ rat lung transplantation was setup. All the rats of control group died in 7d after transplantation, while all survived exceed 7days in the experimental group(DPPⅣenzyme was inhibited specificitily). Comparing with the control group, PIP decreased(P<0.05), PO2 increased (P< 0.05) and W/D decreased (P<0.05), MPO activity and MDA content decreased (P<0.05) in experimental groups. The indexes mentioned above improved in experimental group along with the time and every index approached normal level at the 7 day after operation. Differential proteomics was applied to find the discrepancy protein expression in donor lung tissue after DPPW enzyme was inhibited,2D-PAGE icons at the 1 days after operation were compared between the two set of lung tissues, and 87 discrepancy protein spots were obtained. Multiple discrepancy proteins were identified after mass spectrum peptide finger print map analysis for 15 spots. Amomg the total,9 discrepancy proteins were over-expressed in experimental group while were hypo-expressed or non-expressed in control group.6 were over-expressed in control group.Conclusion:Specific DPPⅣenzyme inhibitor can obviously relieve the IR injury after rat lung transplantation and promote the recovery of lung function. We successfully construct 2-DE icons of rat lung tissue by protein electrophoresis, detect multiple discrepancy protein expression and identify 15 of them. Proteins that can protect lung function or participate in cell signal transduction pathways were detected. ATⅢMay be the substrate of DPPⅣenzyme to protect lung function.更多还原