p97转基因小鼠肺动脉高压动物模型的建立

Pulmonary arterial hypertension is a progressive fatal disease that can lead to right heart failure and death. The understanding mechanism of occlusive vascular remodeling, current treatments only slightly improve the patient's quality of life.One of the main obstacles is the lack of an appropriate spontaneous mouse model of pulmonary hypertension to describe pathophysiological changes in patients with severe pulmonary hypertension. P97/IP3R3/PTEN signaling pathway can regulate the protein quality control of cancer, regulate the ubiquitination and control cell proliferation. Previous studies have shown that the PTEN/AKT/HIF signaling pathway plays a crucial role in the remodeling of pulmonary hypertension vessels. Therefore, the project focuses on the establishment of spontaneous pulmonary hypertension animal model in p97 transgenic mice. The model verifies various pathological phenotypes of pulmonary hypertension disease through animal experiments and cell experiments. The model was evaluated through being compared with the clinical phenotypes of patients with pulmonary hypertension. Gene knockout and transgenic mice models, molecular biological experiments and in vitro pulmonary circulation experiments revealed the role and regulatory mechanism of p97 / PTEN / HIF signaling axis pathway in pulmonary hypertension.This model provides a new model basis for the study of the occurrence, development and complex mechanism of pulmonary hypertension in humans.

肺动脉高压是一种渐进性的致命疾病，可导致右心衰竭而死亡。由于对闭塞性血管重塑的机制了解不足，目前的治疗方法只轻微改善病人的生活质量。主要障碍之一是缺乏适当的自发小鼠肺动脉高压模型来描述肺动脉高压患者的病理生理变化。p97/IP3R3/PTEN信号通路可以调节癌细胞蛋白质方面的质量控制，调节泛素化过程进而控制细胞增殖。前期研究结果表明PTEN/AKT/HIF信号通路在肺动脉高压血管重塑过程中发挥着至关重要的作用。因此本项目致力于建立p97转基因小鼠自发的肺动脉高压动物模型。该模型通过动物实验和细胞实验验证肺动脉高压疾病的各种病理表型，并和人类肺动脉高压患者临床表型做比较，评价其构建效果。结合基因敲除和转基因小鼠模型、分子生物学实验及离体肺循环实验揭示p97/PTEN/HIF信号轴通路在肺动脉高压疾病中的作用及调控机制。为研究人类肺动脉高压的发生、发展及其复杂的机制奠定了新的模型基础。

肺动脉高压（PAH）是一类以肺血管阻力增加和肺动脉压力升高为主要临床症状的呼吸系统疾病。其是一种多因素诱发肺血管疾病，预后差，严重威胁人类健康。F-box蛋白FBXL2结合IP3R3将其靶向泛素化、P97和蛋白酶体介导的降解来限制Ca2+进入线粒体促进细胞存活，表明P97可以促进IP3R3的降解。内质网钙释放通道1,4,5-三磷酸肌醇III型受体（IP3R3）在EC渗透和迁移中发挥关键作用。本项目构建三种PH动物模型、基因敲除鼠PH模型及在体外利用人、大鼠和小鼠细胞实验，探讨IP3R3在PAH发生发展过程中对PAEC的增殖迁移、内皮间充质转化（EndMT）、Ca2+和线粒体功能影响的机制，以及NRF2在调节PAEC和PASMC异常表型中的机制。MCT-PH、SuHx-PH和HPH大鼠模型右心室收缩压、右心肥厚指数和肺血管壁厚度与对照组相比显著升高，提示PAH动物模型建立成功；与对照组相比，IP3R3在MCT-PH、SuHx-PH和HPH大鼠模型肺组织中表达水平都显著上升且主要变化定位为PAEC。持续性低氧诱导的IP3R3（-/-）小鼠右心室收缩压、右心肥厚指数及肺血管壁厚度显著低于HPH小鼠。在人、大鼠及小鼠PAEC中，低氧诱导显著上调IP3R3表达水平；敲低IP3R3后可以显著抑制低氧诱导的大鼠PAEC增殖、迁移及EndMT。EndMT诱导剂TGF-β1显著上调了IP3R3的表达，敲低IP3R3后显著抑制了EndMT和细胞迁移水平。诱导细胞发生EndMT时敲低IP3R3可以下调细胞内Ca2+含量，并且可以抑制EndMT诱导的线粒体损伤，降低线粒体内ROS水平、升高线粒体膜电位和线粒体呼吸链复合物含量。NRF2在PAH动物模型肺组织中的表达水平显著降低。人、大鼠PASMC中，低氧诱导显著上调NRF2的表达，则PAEC中表达下调。PAEC中NRF2的敲除上调ROS水平和EndMT转录因子Snail1蛋白水平。NRF2可以通过调节PDGFR/ERK1/2和mTOR/P70S6K通路来促进细胞增殖，并通过影响MMP2/3/7来加速细胞迁移。综上所述，IP3R3（-/-）小鼠抑制HPH的发生发展，敲低IP3R3可以抑制低氧诱导的PAEC功能异常改变，且敲低IP3R3可以通过Ca2+改善线粒体功能从而抑制EndMT过程，可影响PAH发生发展，将作为预防治疗PAH的潜在靶标。