N-乙酰半胱氨酸对无心跳供体（DCD）肺的保护作用

发布时间：2018-01-16 19:12

  本文关键词：N-乙酰半胱氨酸对无心跳供体（DCD）肺的保护作用　出处：《上海交通大学》2014年硕士论文　论文类型：学位论文

  更多相关文章： 兔 DCD EVLP 肺保护 动物模型 NAC DCD MDA MPO IL-1β 缺血再灌注损伤

【摘要】：肺移植是治疗终末期肺部疾病最为有效的方法，但一直受困于供体的匮乏。近年，来源于无心跳供体(Donation after cardiac death, DCD)的肺移植病例逐年增多，并取得了良好的临床效果。DCD又可分为可控制（或称有准备型）和不可控制（或称无准备型），在供肺的获取和植入过程中，供肺经历了缺血-再灌注损伤（Ischemia-reperfusion injury，IRI）,此损伤可导致器官术后早期原发性移植物功能障碍（Primary graft failure,PGD），这一严重并发症，是导致肺移植术失败及术后患者死亡的重要因素，其病理机制错综复杂，现已经阐明，氧自由基大量产生及白细胞活化，参与了这一损伤病理机制中。 围绕DCD肺的保护，出现了“常温体外供肺灌注”技术，即离体肺灌注（Ex vivolung perfusion,EVLP）。EVLP的应用，有助于降低PGD，提高DCD肺的利用率，减轻肺水肿，降低IRI对肺功能的损害，并可作为供肺评估和术前预处理的平台。 已经有研究证明N-乙酰半胱氨酸（N-acetylcysteine,NAC）在有心跳供体肺移植中能抑制中性粒细胞生成，从而抑制通过ROS/RNS通路激活的NF-kB，避免脂质过氧化反应的发生引起的细胞调亡,同时，在体内激活鸟苷酸环化酶，升高血浆环磷酸鸟苷酸（cGMP）水平，增加体内NO的生物作用，对白细胞介素、血小板活化因子等产生抑制作用，从而缓解肺缺血-再灌注损伤，迄今为止，NAC对DCD供肺是否同样拥有显著的保护作用，尚不清楚，有待进一步研究。 本研究通过建立兔离体肺灌注（Ex vivo lung perfusion,EVLP）模型，随后在该模型上，研究N-乙酰半胱氨酸对无心跳供体肺组织的保护作用。 本研究结果显示，兔DCD肺模型及EVLP灌注模型具有较好的可靠性和可重复性，可以长时间获取呼吸功能的参数，包括监测肺顺应性和动脉血气。不仅可对肺移植后的效果进行预评估（评估平台），还可为药物（NAC）对DCD肺保护的研究，提供一个良好的研究平台; NAC能显著提高肺的GSH含量，从而抑制肺氧自由基的升高；同时抑制炎性物质的释放，降低髓过氧化物酶、丙二醛、il-1β含量，降低气道阻力，对于dcd肺同样具有良好的保护作用。综上所述，兔evlp能够作为体外肺灌注研究的可靠模型，同时nac能够减轻肺缺血-再灌注损伤（iri），为无心跳供体肺保护提供了一种新的保护手段。 第一部分兔无心跳供体(DCD)肺模型及离体肺灌注（EVLP）模型的建立 目的：构建一个离体肺灌注（EVLP）模型，并将缺血诱导的兔DCD肺进行离体灌注和通气，探讨该EVLP模型效果。 方法：成年（15-20周龄）新西兰大白兔（雄性），体重2-2.5kg共6只，麻醉开胸后，，兔的左心室心尖插入引流管，放血至储血袋中备用。心跳停止后静置1h，肺动脉与左心房分别灌注Perfadex保护液，灌注完毕后，4℃Perfadex液冷保存3h后取出，固定于EVLP装置上，同时，储血袋中血注入氧合器中，充95％N和5％CO2之混合气体，进行去氧合。蠕动泵将38℃去氧合血，泵至兔的主肺动脉进行灌注；经左心房溢出的回流之氧合血，流入储心肺罐中，蠕动泵将其重新泵至氧合器中，再次进行去氧合。 结果：除1例因肺动脉肺动脉误伤撕裂后，出血量大，被迫中断实验外，其余动物均顺利完成实验。灌注90min后肺组织少许肿胀，均无肺梗死病灶出血，灌注结束后，肺气道峰压较灌注前上升，肺含水量无明显增多。光镜观察，肺组织细胞结构基本清晰完整。 结论：本实验采用的方法，建立起来的动物模型稳定、可重复性好。 第二部分N-乙酰半胱氨酸对无心跳供体(DCD)肺保护作用的实验研究 目的：探讨NAC对DCD供肺能否进一步提供保护作用。 方法：成年（15-20周龄）新西兰大白兔（雄性），体重2-2.5kg，根据灌洗液（F）、保存液（P）是否加入NA（C150mg/kg）将大白兔随机分为3组：组1为对照组（F+PerfadexP+Perfadex），组2（F+Perfadex+NAC P+Perfadex）、组3(F+Perfadex+NACP+Perfadex+NAC)为加入NAC实验组(每组n=6)。缺血诱导供心停跳，停跳后继续热缺血（室温下静置）60min之后阻断主动脉，上、下腔静脉，经主肺动脉顺行及左心房逆行灌注灌洗液（F）。灌注完毕后，切取供心肺，4℃保存液（P）保存3小时后取出将其固定在EVLP装置上，去氧合38℃血持续灌注肺动脉，呼吸机通气，观察90min,结束灌注。灌注结束后，切取肺组织，测定肺组织含水率, MDA、MPO水平，IL-1β检测，蛋白浓度检测，肺组织光镜下观察。 结果：对照组在肺组织含水率、气道峰压、MDA、MPO、IL-1β等检测中均显著高于实验组，差异有统计学意义（P0.05），对照组在氧分压、GSH检测中低于实验组（P0.05），蛋白浓度检测两组无显著性差异（P0.05），光镜下观察对照组较实验组存在更明显结构组织破坏及炎性细胞浸润。实验组之间各项指标比较均无显著性差异（P0.05）。 结论： NAC对DCD供肺具有保护作用，可降低肺组织的移植炎症细胞反应，减轻肺的氧自由基破坏，降低缺血再灌注损伤，为DCD供肺的保存提供了一种新的途径。
[Abstract]:Lung transplantation is the most effective method for the treatment of end stage lung disease, but suffers from the lack of donor. In recent years, from non heart beating donor (Donation after, cardiac death, DCD) of lung transplantation cases increased year by year, and achieved good clinical effect of.DCD can be divided into control (or ready type) and can not control (or to), the donor lung acquisition and implantation process, donor lung experienced ischemia reperfusion injury (Ischemia-reperfusion, injury, IRI), this can lead to organ damage early postoperative primary graft dysfunction (Primary graft, failure, PGD), which is a serious complication that is an important factor leading to death in patients with lung transplantation failure and after surgery, the pathological mechanism has been elucidated, perplexing, oxygen free radicals and leukocyte activation, involved in the pathological mechanism.
Around the protection of lung DCD, a pulmonary perfusion technique for in vitro at room temperature, the isolated lung perfusion (Ex vivolung, perfusion, EVLP).EVLP applications, helps to reduce PGD, improve the utilization rate of DCD in the lung, reduce pulmonary edema, reduce IRI damage of lung function, and can be used as a pretreatment for lung assessment and preoperative platform.
It has been proved that N- acetylcysteine (N-acetylcysteine, NAC) in the heart beating donor can inhibit neutrophil generation of lung transplantation, thereby inhibiting the activation of ROS/RNS pathway by NF-kB, to avoid the occurrence of lipid peroxidation induced cell apoptosis, and the activation of guanylate cyclase in the body, the increase of plasma cyclic guanosine acid (cGMP) level, increase the biological role of NO, interleukin, platelet activating factor produced inhibitory effect, thereby alleviating lung ischemia-reperfusion injury, so far, NAC DCD of donor lung whether also has a significant protective effect, is not clear, need to be further studied.
In this study, we established a rabbit model of Ex vivo lung perfusion (EVLP), and then studied the protective effect of N- acetylcysteine on the lung tissue of non heart beating donors.
The results of this study show that the rabbit DCD lung model and EVLP perfusion model has good reliability and repeatability, parameters can be obtained for long time monitoring of the respiratory function, including lung compliance and arterial blood gas. Not only can evaluate the effect after lung transplantation (evaluation platform), but also for drug research (NAC) the DCD of lung protection, provide a good research platform; NAC can significantly increase the content of GSH in lung, lung increased to inhibit oxygen free radicals; at the same time, inhibition of inflammatory substances release, decreased myeloperoxidase, malondialdehyde, IL-1 beta content, reduce airway resistance, also has good protective effect on lung DCD. In summary rabbit, evlp can be used as a reliable model in vitro lung perfusion, while NAC can reduce lung ischemia-reperfusion injury (IRI), a non heart beating donor lung protection provides a new means of protection.
The first part of the rabbit model of non heartbeat donor (DCD) lung and the establishment of EVLP model in vitro
Objective: to construct an isolated lung perfusion (EVLP) model, and to investigate the effect of this EVLP model by perfusion and ventilation in the DCD lung of rabbit induced by ischemia.
Methods: adult (15-20 weeks) New Zealand white rabbits (male), a total of 6 2-2.5kg body weight, anesthesia after thoracotomy, left ventricular apical rabbit is inserted into the drainage tube, spare blood to a blood storage bag. After cardiac arrest in the static 1H, pulmonary artery and left atrium were perfused with Perfadex protective liquid infusion is completed after 4 DEG Perfadex solution cold preservation after 3H removed, fixed on the EVLP device, at the same time, blood storage bag of blood into the oxygenator, mixed gas filled 95%N and 5%CO2, were deoxygenated. Peristaltic pump 38 DEG C deoxygenated blood pump, the rabbit to the main pulmonary artery perfusion; after returning to the left the atrial overflow of oxygenated blood, heart and lung into the storage tank, the peristaltic pump pump to re oxygenation, again deoxygenated.
Results: except for 1 cases of pulmonary injury after tearing, bleeding, was forced to interrupt the experiment, the animal experiments were completed successfully. The lung tissue swelling a little after 90min, there were no hemorrhagic pulmonary infarction lesions after reperfusion, lung perfusion was increased before the peak airway pressure, lung water content was significantly increased. Light microscope, structure of lung tissue cells is clear and complete.
Conclusion: the animal model established in this experiment is stable and reproducible.
Experimental study on the protective effect of N- acetyl cysteine on lung protection of non heartbeat donor (DCD) in second parts
Objective: To investigate whether NAC can provide a further protective effect on DCD donor lung.
鏂规硶锛氭垚骞

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