肺泡上皮细胞氧脱适应在低氧性肺动脉高压中的作用及分子机制

Hypoxic pulmonary hypertension (HPH) is a pathological process characterized by pulmonary artery contraction and remodeling, which pathogenesis have no breakthrough yet. Alveolar epithelial cells (AEC) first perceive hypoxia in the lungs, but their role in HPH has long been neglected. Existing facts indicate that only alveolar hypoxia can cause HPH. Our study results showed that both pulmonary artery and bronchial artery belonging to the systemic circulation occured structural remodeling located in the lung hypoxic microenvironment. Thus, the subject proposes "microenvironment theory" for pathogenesis of pulmonary hypertension. Hypoxia makes the AEC decondition to oxygen. The oxygen-deconditioned AEC mainly occurs changes in Redox pathway, which is the source of reactive oxygen species (ROS) in the pulmonary microenvironment caused by hypoxia. It is lung hypoxic microenvironment that promotes pathological changes of vessels located in lung. The project should establish animal models of unilateral hypoxic pulmonary hypertension in rats with bronchial stenosis, compared to the healthy side lung to investigate the role of changed lung microenvironment in HPH developing. The study will also investigate the effects of oxygen-deconditioned AEC on the pulmonary artery smooth muscle cells (PASMCs) proliferation, apoptosis and contraction using cellular model of co-cultured AEC and PASMCs. The study will further explore the dynamic changes and pathophysiological significance of ROS and thioredoxin (Trx) systems in lung microenvironment in the development of HPH. It should provide a new theoretical basis and clues for the pathogenesis and prevention of HPH.

低氧性肺动脉高压（HPH）以肺小动脉收缩和结构重建为病理变化特征。肺部低氧时，肺泡上皮细胞（AEC）首先感知，但其在HPH中的作用常被忽视。已知只有肺泡缺氧才能引起HPH，我们的研究结果显示低氧时位于肺微环境中的肺动脉和属于体循环的支气管动脉都会发生结构重建。因此提出假设：低氧使AEC发生氧脱适应，影响其氧化还原通路，引起肺微环境氧化还原状态改变，使处于肺微环境中的血管发生病变，即HPH发生的"肺微环境学说"。本项目拟采用大鼠肺支气管狭窄术，建立单侧HPH动物模型，以健侧肺脏为对照，研究肺微环境改变在HPH发生中的作用；采用共培养细胞模型，探讨AEC氧脱适应对肺动脉平滑肌细胞（PASMCs）增殖、凋亡和收缩的影响；利用药理学、分子生物学等手段，探讨AEC主导的肺微环境中ROS、硫氧还蛋白系统在HPH发生发展中的动态变化及其病理生理学意义，为HPH的发病机制和防治提供新的理论基础和线索。

低氧性肺动脉高压（HPH)以肺小动脉收缩和结构重建为重要病理变化，肺部低氧时，肺胞上皮细胞最先感知，它的变化对HPH有何影响知之甚少。本项目提出假设：低氧使生理状态下处于相对较高氧浓度的肺泡上皮细胞发生氧脱适应性变化，影响其氧化还原通路，引起肺微环境氧化还原状态改变，使处于肺环境中的血管发生病理变化，即HPH发生的“微环境学说”。本研究项目通过在体单侧支气管狭窄肺动脉高压动物模型和体外肺泡上皮细胞低氧条件培养液以及肺泡上皮细胞与血管平滑肌细胞（VSMCs，包括肺动脉和主动脉平滑肌细胞，PASMCs、AASMCs）同培养技术，采用基因测序、RT-PCR，Western blot、流式细胞术、同聚焦显微镜以及免疫荧光等技术测定了肺动脉压力、肺血管壁厚度、右心室指数、细胞增殖、凋亡以及ROS、H2O2等指标的变化，探讨肺胞上皮细胞在HPH发生发展中的作用及其机制。研究结果表明低氧可使肺泡上皮细胞表型发生改变，与常氧组比较，低氧组差异表达基因664个，其中429个基因上调，235个基因下调，与低氧性肺动脉高压（HPH）相关的主要包括三个方面：氧化应激反应系统、血管紧张素系统和IL-6系统。动物和细胞培养均表明：低氧时肺部组织和肺泡上皮细胞内ROS及培养液中H2O2升高，NO含量减少。实验还证实：常氧条件下的肺胞上皮细胞或其培养液能够抑制常氧或低氧培养PASMCs和AASMC的增殖，促进其凋亡，但低氧暴露的肺泡上皮细胞或其低氧条件培养液却能够促进无论是低氧或常氧培养的PASMCs和AASMCs增殖，减弱其促凋亡作用；阐明了肺泡上皮细胞在低氧条件下是通过ROS/H2O2途径促进HPH发生发展的。上述结果具有重要的理论意义和临床价值，其一：丰富或完善肺部血流量大但肺循环阻力低的传统理论解释，即正常生理状态下，肺循环阻力低除了肺血管床丰富外，肺胞上皮细胞也发挥重要作用；其二，由于低氧影响了肺胞上皮细胞的功能，使肺部微环境中ROS/H2O2增加，促进肺部血管收缩、增殖，为HPH发生发展的“微环境学说”提供了科学依据，为HPH的防治提供了明确的靶点。