氧合血体外持续灌注对供肺保护的动物实验研究

The lung preservation is the key to success of the lung transplantation, ischemia-reperfusion-induced lung injury remains a significant cause of early morbidity and mortality after lung transplantation.. The disadvantage of "Classic static preservation in deep temperature and preservative solution" includes oxidative Stress and the inhibition of cellular metabolism induced by protective hypothermia obviating the possibility of substantial reparative processes ,and so on.Therefore,donor lungs can only tolerate up to 4～8 h of aerobic cold ischemia.Because of the influences of ventilator,"ex vivo lung perfusion assisted with ventilator" is not suitable for remote transportation, and is only used in initiate reparative processes and real-time quality assessment of high-risk donor lungs. Although the lung has two groups of pulmonary and bronchial blood circulation, there are capillary net anastomosed between the two circulations.SO, we present a hypothesis that without the application of ventilator,ex vivo oxygenated blood by continuous perfusion in pulmonary circulation could reach the interstitium and the alveolar space of donor lung,and provide oxygen supply. It has been preliminarily proved during the initial research. To confirm further this hypothesis, in this porcine animal study, we observe the protective effect of ex vivo oxygenated blood continuous perfusion to prevent donor-lung injury,by evaluation of the lung injury,and apoptosis,and so on,during postoperative 72h. This project will present a new approach of lung preservation,which is suitable for remote transportation.

供肺保存是肺移植成功的关键之一，缺血-再灌注诱导的肺损伤仍是肺移植术后早期死亡的重要原因。经典"深低温静态保存法"因氧化应激及深低温细胞代谢抑制丧失自我修复能力等不足,供肺仅能保存4-8小时；"结合呼吸机通气的供肺体外灌注技术"因离体肺氧合仍依靠呼吸机，故此方法不适于长时间远距离运输供体，而主要用于边缘供肺功能修复与肺功能评估。肺具有肺动静脉（功能血管）和支气管动静脉（营养血管），但两组血管间有毛细血管网吻合；我们提出假说"不使用呼吸机、氧合血经肺动脉体外灌注保存供肺,氧合血通过毛细血管网维持组织氧供及营养，减轻缺血-再灌注损伤"，已在前期研究中得到初步证实。为进一步证明这一假说，本实验拟以广西巴马小型猪为实验对象，"氧合血体外持续灌注供肺（不使用呼吸机）"后行肺移植，采用肺损伤及细胞凋亡等研究指标，观察术后72h供肺缺血-再灌注损伤程度,为供肺保存提供一种适于远距离运输的灌注技术和方法。

【摘要】: 目的 体内生理状态评估氧合血体外持续灌注对离体供肺的保护作用。方法 将细胞外保护液(Celsior液)单次经肺动静脉灌注后的猪肺,分为Ⅰ组（深低温保存组,n=8），Ⅱ组（氧合血体外持续灌注保存组,n=8)，Ⅲ组（多伦多离体肺体外灌注技术保存组,n=8），保存8h后，将组猪肺移植至受体猪体内，连续监测移植后72 h，研究三组移植后0h、12h、24h、48h、72h相关指标。肺功能指标：肺通气阻力(LR)、左下肺静脉血氧分压(LIPV-PO2)及肺含水量；肺损伤检测：透射电镜以判断凋亡的发生及细胞类型，原位末端标记(TUNEL)法检测细胞凋亡计数。结果 （1）术后相同时间点Ⅱ组及Ⅲ组LR及肺含水量显著低于I组（均P＜0.05)、LIPV-PO2显著高于Ⅰ组（均P＜0.05)，Ⅱ组及Ⅲ组肺功能指标术后无显著差异（均P＞0.05)；（2）各组肺移植术后LR及肺含水量呈升高-降低曲线，峰值均出现于术后12h;LIPV-PO2移植术后呈下降-上升曲线，谷底值亦均出现于术后12h ；(3)移植术后三组均可见以Ⅱ型肺泡上皮细胞为主的凋亡细胞，凋亡高峰期均出现在术后12h; Ⅱ组及Ⅲ组在术后24h即迅速进入以晚期凋亡细胞为主的凋亡消退期，Ⅰ组在术后72h才进入凋亡消退期； (4) Tunel染色凋亡细胞计数Ⅱ组、Ⅲ组移植术后各时间点肺泡细胞凋亡率显著低于Ⅰ组（均P＜0.05）；各组内比较，Ⅰ组肺泡细胞凋亡率呈上升-下降曲线，移植术后各时间点显著高于供肺保存前（（均P<0．05)），峰值出现在移植术后12h；Ⅱ组、Ⅲ组于移植术12h、24h后肺泡细胞凋亡率显著高于供肺保存前(Ⅱ组P值分别为0.00,0.01； Ⅲ组P值分别为0.01，0.02)。结论 (1) 体外持续灌注氧合血保存供肺能有效减轻缺血-再灌注损伤，保护肺功能，其供肺保存效果明显优于“深低温静态保存”方式。此方法通过通过降低肺泡细胞凋亡水平，从而减少缺血再灌注损伤，改善供肺的保护。(2) 体外持续灌注氧合血保存的供肺仍存在肺泡细胞凋亡的发生、发展及一定程度的缺血-再灌注损伤，启动机制有待进一步研究。