本文關(guān)鍵詞：N-乙酰半胱氨酸對(duì)無(wú)心跳供體（DCD）肺的保護(hù)作用　出處：《上海交通大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 兔 DCD EVLP 肺保護(hù) 動(dòng)物模型 NAC DCD MDA MPO IL-1β 缺血再灌注損傷

【摘要】：肺移植是治療終末期肺部疾病最為有效的方法，但一直受困于供體的匱乏。近年，來(lái)源于無(wú)心跳供體(Donation after cardiac death, DCD)的肺移植病例逐年增多，并取得了良好的臨床效果。DCD又可分為可控制（或稱有準(zhǔn)備型）和不可控制（或稱無(wú)準(zhǔn)備型），在供肺的獲取和植入過(guò)程中，供肺經(jīng)歷了缺血-再灌注損傷（Ischemia-reperfusion injury，IRI）,此損傷可導(dǎo)致器官術(shù)后早期原發(fā)性移植物功能障礙（Primary graft failure,PGD），這一嚴(yán)重并發(fā)癥，是導(dǎo)致肺移植術(shù)失敗及術(shù)后患者死亡的重要因素，其病理機(jī)制錯(cuò)綜復(fù)雜，現(xiàn)已經(jīng)闡明，氧自由基大量產(chǎn)生及白細(xì)胞活化，參與了這一損傷病理機(jī)制中。 圍繞DCD肺的保護(hù)，出現(xiàn)了“常溫體外供肺灌注”技術(shù)，即離體肺灌注（Ex vivolung perfusion,EVLP）。EVLP的應(yīng)用，有助于降低PGD，提高DCD肺的利用率，減輕肺水腫，降低IRI對(duì)肺功能的損害，并可作為供肺評(píng)估和術(shù)前預(yù)處理的平臺(tái)。 已經(jīng)有研究證明N-乙酰半胱氨酸（N-acetylcysteine,NAC）在有心跳供體肺移植中能抑制中性粒細(xì)胞生成，從而抑制通過(guò)ROS/RNS通路激活的NF-kB，避免脂質(zhì)過(guò)氧化反應(yīng)的發(fā)生引起的細(xì)胞調(diào)亡,同時(shí)，在體內(nèi)激活鳥(niǎo)苷酸環(huán)化酶，升高血漿環(huán)磷酸鳥(niǎo)苷酸（cGMP）水平，增加體內(nèi)NO的生物作用，對(duì)白細(xì)胞介素、血小板活化因子等產(chǎn)生抑制作用，從而緩解肺缺血-再灌注損傷，迄今為止，NAC對(duì)DCD供肺是否同樣擁有顯著的保護(hù)作用，尚不清楚，有待進(jìn)一步研究。 本研究通過(guò)建立兔離體肺灌注（Ex vivo lung perfusion,EVLP）模型，隨后在該模型上，研究N-乙酰半胱氨酸對(duì)無(wú)心跳供體肺組織的保護(hù)作用。 本研究結(jié)果顯示，兔DCD肺模型及EVLP灌注模型具有較好的可靠性和可重復(fù)性，可以長(zhǎng)時(shí)間獲取呼吸功能的參數(shù)，包括監(jiān)測(cè)肺順應(yīng)性和動(dòng)脈血?dú)�。不僅可對(duì)肺移植后的效果進(jìn)行預(yù)評(píng)估（評(píng)估平臺(tái)），還可為藥物（NAC）對(duì)DCD肺保護(hù)的研究，提供一個(gè)良好的研究平臺(tái); NAC能顯著提高肺的GSH含量，從而抑制肺氧自由基的升高；同時(shí)抑制炎性物質(zhì)的釋放，降低髓過(guò)氧化物酶、丙二醛、il-1β含量，降低氣道阻力，對(duì)于dcd肺同樣具有良好的保護(hù)作用。綜上所述，兔evlp能夠作為體外肺灌注研究的可靠模型，同時(shí)nac能夠減輕肺缺血-再灌注損傷（iri），為無(wú)心跳供體肺保護(hù)提供了一種新的保護(hù)手段。 第一部分兔無(wú)心跳供體(DCD)肺模型及離體肺灌注（EVLP）模型的建立 目的：構(gòu)建一個(gè)離體肺灌注（EVLP）模型，并將缺血誘導(dǎo)的兔DCD肺進(jìn)行離體灌注和通氣，探討該EVLP模型效果。 方法：成年（15-20周齡）新西蘭大白兔（雄性），體重2-2.5kg共6只，麻醉開(kāi)胸后，，兔的左心室心尖插入引流管，放血至儲(chǔ)血袋中備用。心跳停止后靜置1h，肺動(dòng)脈與左心房分別灌注Perfadex保護(hù)液，灌注完畢后，4℃Perfadex液冷保存3h后取出，固定于EVLP裝置上，同時(shí)，儲(chǔ)血袋中血注入氧合器中，充95％N和5％CO2之混合氣體，進(jìn)行去氧合。蠕動(dòng)泵將38℃去氧合血，泵至兔的主肺動(dòng)脈進(jìn)行灌注；經(jīng)左心房溢出的回流之氧合血，流入儲(chǔ)心肺罐中，蠕動(dòng)泵將其重新泵至氧合器中，再次進(jìn)行去氧合。 結(jié)果：除1例因肺動(dòng)脈肺動(dòng)脈誤傷撕裂后，出血量大，被迫中斷實(shí)驗(yàn)外，其余動(dòng)物均順利完成實(shí)驗(yàn)。灌注90min后肺組織少許腫脹，均無(wú)肺梗死病灶出血，灌注結(jié)束后，肺氣道峰壓較灌注前上升，肺含水量無(wú)明顯增多。光鏡觀察，肺組織細(xì)胞結(jié)構(gòu)基本清晰完整。 結(jié)論：本實(shí)驗(yàn)采用的方法，建立起來(lái)的動(dòng)物模型穩(wěn)定、可重復(fù)性好。 第二部分N-乙酰半胱氨酸對(duì)無(wú)心跳供體(DCD)肺保護(hù)作用的實(shí)驗(yàn)研究 目的：探討NAC對(duì)DCD供肺能否進(jìn)一步提供保護(hù)作用。 方法：成年（15-20周齡）新西蘭大白兔（雄性），體重2-2.5kg，根據(jù)灌洗液（F）、保存液（P）是否加入NA（C150mg/kg）將大白兔隨機(jī)分為3組：組1為對(duì)照組（F+PerfadexP+Perfadex），組2（F+Perfadex+NAC P+Perfadex）、組3(F+Perfadex+NACP+Perfadex+NAC)為加入NAC實(shí)驗(yàn)組(每組n=6)。缺血誘導(dǎo)供心停跳，停跳后繼續(xù)熱缺血（室溫下靜置）60min之后阻斷主動(dòng)脈，上、下腔靜脈，經(jīng)主肺動(dòng)脈順行及左心房逆行灌注灌洗液（F）。灌注完畢后，切取供心肺，4℃保存液（P）保存3小時(shí)后取出將其固定在EVLP裝置上，去氧合38℃血持續(xù)灌注肺動(dòng)脈，呼吸機(jī)通氣，觀察90min,結(jié)束灌注。灌注結(jié)束后，切取肺組織，測(cè)定肺組織含水率, MDA、MPO水平，IL-1β檢測(cè)，蛋白濃度檢測(cè)，肺組織光鏡下觀察。 結(jié)果：對(duì)照組在肺組織含水率、氣道峰壓、MDA、MPO、IL-1β等檢測(cè)中均顯著高于實(shí)驗(yàn)組，差異有統(tǒng)計(jì)學(xué)意義（P0.05），對(duì)照組在氧分壓、GSH檢測(cè)中低于實(shí)驗(yàn)組（P0.05），蛋白濃度檢測(cè)兩組無(wú)顯著性差異（P0.05），光鏡下觀察對(duì)照組較實(shí)驗(yàn)組存在更明顯結(jié)構(gòu)組織破壞及炎性細(xì)胞浸潤(rùn)。實(shí)驗(yàn)組之間各項(xiàng)指標(biāo)比較均無(wú)顯著性差異（P0.05）。 結(jié)論： NAC對(duì)DCD供肺具有保護(hù)作用，可降低肺組織的移植炎癥細(xì)胞反應(yīng)，減輕肺的氧自由基破壞，降低缺血再灌注損傷，為DCD供肺的保存提供了一種新的途徑。
[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.
鏂規(guī)硶錛氭垚騫

本文編號(hào)：1434431