体外肺灌注（EVLP）结合药物干预改善大鼠肺移植后缺血再灌注损伤的作用和机制

Lung transplantation is the only curative therapy for end-stage lung disease. Nevertheless, the development of such modality is severely restrained, due to the quality and the quantity insufficiency of the eligible donor lung offered. It is believed that the primary graft dysfunction, characterized by the onset of severe ischemia reperfusion injury, contributes to the increased mortality post transplant. The emerging ex-vivo lung perfusion sets a modern lung graft preservation approach in order to elevate the acceptance rate of donor organ, but its mechanism in ameliorating the reperfusion injury remains unclear. The applicant has previously shown that pharmacological inhibition of free radicals and down-stream inflammatory products during EVLP leads to an improved pulmonary physiology prior to transplant. We hypothesize that multiple pharmacological interventions targeting the oxidative/nitrosative stress during EVLP could mitigate the reperfusion injury following transplants. The current project aims to explore a new treatment strategy against IRI during EVLP, and to identify new therapeutic targets by uncovering the transcriptome of lungs with IRI. Our major objectives are: 1) To establish a pharmacological EVLP platform, and to screen the efficacy of several therapeutic candidates in treating damaged donor lungs. 2) To examine the expression of damage-associated molecular patterns (DAMPs) during IRI to determine whether they can serve as functional biomarkers of donor lungs. 3) To identify new molecular pathways or genes that are altered in lungs with IRI by RNA-Seq. This study will shed important new lights to better understanding the effect of therapeutic EVLP as well as the mechanism of IRI following lung transplantation.

肺移植是治疗终末期肺疾病的唯一有效手段，但受限于供肺质量和数量，临床肺移植的发展和推广一直较缓慢。而供肺缺血再灌注损伤所致的原发性移植物失功是造成肺移植术后早期较高死亡率的重要原因。近年发展起来的常温体外肺灌注(EVLP)技术一定程度上扩大了供肺利用率，但其缓解移植后再灌注损伤的作用机制仍未阐明。申请者前期研究中发现在EVLP中阻断损伤性氮氧自由基生成、抑制其下游炎症通路可显著改善供肺质量。基于该发现，本项目提出假设：在EVLP条件下，联合药物性干预氧化或硝基化应激通路，有望缓解缺血供肺移植后再灌注损伤的发生。本项目拟建立大鼠损伤供肺的多种药物治疗性EVLP模型及肺移植模型；并在此基础上定量分析肺移植再灌注后损伤相关的分子模式和移植物失功的相互关系，进一步对移植供肺实质细胞进行全转录组测序，扩充EVLP修复损伤的生物学评价指标，探索上述联合干预改善供肺移植后再灌注损伤的作用及机理。

肺移植是治疗部分终末期肺疾病的唯一有效手段。常温体外肺灌注技术（EVLP）可扩大供肺利用率，但其缓解移植后缺血再灌注损伤的机制尚不明确。本研究首先建立了不同时长冷缺血和热缺血保存条件的肺损伤模型，证实大鼠损伤供肺可在EVLP中进行生理性评估和修复。发现一小时内热缺血供肺在EVLP中其损伤可逆；而两小时热缺血损伤供肺其肺顺应性、肺血管阻力、氧合指数等在EVLP中明显恶化，产生不可逆损伤。研究进一步确立了氧化应激和硝基化应激是供肺缺血再灌注损伤的核心机制。发现蛋白质羰基，乳酸脱氢酶，3-硝基酪氨酸等产物在不同温度造成的缺血肺损伤中起关键作用。常温EVLP后，两小时热缺血损伤供肺其肺泡灌洗液或肺组织中上述产物较一小时热缺血或冷缺血损伤肺显著增加。该结果揭示了氧化应激和硝基化应激是缺血再灌注损伤的重要活化通路，同时也为干预调控氧化应激损伤提供可能。研究者通过在EVLP转流过程中加入3-氨基苯甲酰胺（3-AB）对供肺热缺血再灌注中间产物聚腺苷二磷酸-核糖聚合酶（PARP）进行药物性阻断，证实了EVLP结合药物性干预能改善供肺顺应性、气道峰压、肺血管阻力和氧合能力。进一步探索了上述预处理供肺在单肺移植开放循环后其顺应性和氧合能力的改善情况。课题还测试了EVLP联合体外抗生素治疗可减少大鼠急性肺炎链球菌感染供肺和EVLP系统细菌载量的疗效，但对肺功能改善不明显。初步说明EVLP作为修复平台对供肺缺血再灌注损伤进行预干预的潜力。在本基金支持下，研究者对131例出院新冠确诊患者进行了随访，发现个别病人因核酸转阳再次入院但其密切接触者未感染新冠。此外，对22例新冠核酸持续阳性大于发病后50天的患者进行了鼻炎分泌物病毒分离测序、免疫功能和中和抗体测定。发现分泌γ-干扰素的NK细胞活性降低和中和抗体滴度降低是造成新冠核酸长阳的原因，但其病毒载量和传染性都不高。