冷缺血期氢气膨肺对移植肺脏功能的影响及作用机制研究

Currently, the protective methods for lung donor was predominated by hypothermia, improvement of the preservation solution and the lung perfusion, and it is urgent to explore new methods. Oxidative stress and inflammation were the main cause of lung injury during the cold ischemic phase. Hydrogen had protective effect in several kinds of lung injury, we also found that hydrogen reduced the oxidative stress and inflammation response of lung graft when applied in the brain death donor or cold ischemia phase. But the effect of hydrogen on cold ischemic lung injury still need further research, and the mechanism of protective effect of hydrogen on lung transplant-related lung injury is unclear. This project intends to observe the effect of lung inflation with hydrogen during the cold ischemic phase on cold ischemia lung injury and lung graft function. We will observe the effects of hydrogen on oxidative stress and inflammatory response at the cellular level through pulmonary microvascular endothelial cells (PMVECs) hypoxia/reoxygenation model, and RNAi technology and related molecular biology techniques; observe the role of p38MAPK, nuclear factor E2-related factor 2 (Nrf2) signaling pathway, and mitochondria mediated apoptosis in the phenomenon of hydrogen exert therapeutic effect on lung injury during the cold ischemia phase and lung graft. In this study, we will explore a new protective method for cold ischemia lung injury through lung inflation with hydrogen, and explore the molecular mechanism of hydrogen on lung transplantation related lung injury, and provide the theoretical basis for hydrogen application in the lung transplantation.

目前肺移植供体保护以低温、肺保存液和肺脏灌注方法的改进为主，亟待探索新方法。氧化应激和炎症反应是冷缺血肺损伤的主要原因。氢气对多种肺损伤具有保护作用，我们亦发现氢气应用于供体冷缺血期和脑死亡后均可改善移植后肺脏功能。但氢气对冷缺血期肺损伤的影响尚需深入研究，其作用机制尚不清楚。本课题拟进一步观察冷缺血期氢气膨肺对供体肺损伤和移植物功能的影响，通过肺微血管内皮细胞（PMVECs）缺氧/复氧模型，采用基因沉默技术及相关分子生物学技术，检测p38MAPK炎症信号通路、核因子E2相关因子2（Nrf2）氧化应激信号通路、及线粒体介导的细胞凋亡在氢气肺移植供体保护中的作用，探讨氢气在肺移植供体保护中抗炎症、抗氧化和抗凋亡机制。本研究通过首创的冷缺血期氢气膨肺技术，探索肺移植供体保护新方法，并探讨氢气在肺移植相关肺损伤中的亚细胞和分子作用机制，为氢气在肺移植过程中的应用提供理论基础。

项目背景：我们前期的研究发现氢气通气可改善由缺血再灌注引发的肺损伤，然而冷缺血期氢气膨肺对肺移植供体的保护效应需要进一步证实，氢气的作用机制需要深入探讨。研究内容：通过动物实验观察冷缺血期使用 3%氢气膨肺对供体肺功能、炎症反应、氧化应激、能量代谢与细胞坏死或凋亡的影响，并进一步探索氢气膨肺对移植后肺功能的保护作用。通过离体肺微血管内皮细胞缺氧/复氧模型研究氢气对PMVECs氧化应激、炎症与细胞凋亡的保护作用；并通过基因干扰技术沉默p38MAPK等基因的表达，探讨氢气的肺保护作用机制。研究结果：氢气膨肺能够改善冷缺血期的组织损伤，包括减少粒细胞浸润与透明膜形成，降低炎症因子（IL-6、IL-10和TNF-α）水平与氧化应激反应，保护细胞凋亡与线粒体结构等。通过对移植后肺组织进行检测，我们发现氢气可同样减轻再灌注引发的炎症反应与氧化应激，而且通过对线粒体结构与复合物功能的保护改善细胞能量代谢。通过PMVECs模拟肺移植过程，我们发现氢气的抗炎、抗氧化与抗凋亡作用主要是通过激活p38MAPK与Nrf2通路来实现的；而且通过腺病毒基因沉默（RNAi）技术，我们发现氢气的保护效应可被Nrf2转录基因的沉默减弱，证实氢气可能通过Nrf2通路来实现肺移植的保护效应。