ROS/HIF-1α/Skp2信号通路在低氧性平滑肌细胞增殖反应阈形成及血管重构中的作用及机制研究

Pulmonary vascular remodeling especially the proliferation of pulmonary vascular smooth muscle cell is the characteristic pathological changes of the hypoxic pulmonary hypertension (HPH), which underlying exact mechanism is still not clear. Our experiment results showed that when vessel smooth muscle cell (VSMCs) were exposed to 10% O2, the proliferation occurred only in cultured.pulmonary VSMCs, but not systemic VSMCs; When exposed to 3% O2, both cultured pulmonary and systemic VSMCs proliferated; But when exposed to 1% O2, the proliferation of both cultured pulmonary and systemic VSMCs were suppressed. The results suggest that there is a different sensitivity to hypoxia induced-proliferation between systemic and pulmonary VSMCs. Therefore, we hypothesis that the different “VSMCs hypoxic proliferative response threshold” exists in the pulmonary and the systemic vessels to explain the mechanism of alveolar hypoxia occurs only in lung VSMCs proliferation and pulmonary hypertension. In view of the role of ROS，HIF-1α and Skp2 in hypoxia and VSMCs proliferation, the present project is intended to investigate the effects of different oxygen concentrations on the systemic and pulmonary circulation, the proliferation and the apoptosis of VSMCs, and to identify the hypoxic proliferative response threshold of VSMCs. We will also Study the effect of ROS, HIF-1α，Skp2/P27 pathway on the proliferation of VSMC and explore the key molecules determining the hypoxic proliferative response threshold and its mechanisms in the development of hypoxic vessel remodeling and proliferation of VSMCs. The results may afford new theory for the mechanisms of the HPH and new target for treatment of hypoxic proliferation of VSMCs.

肺血管重构特别是肺血管平滑肌细胞增殖是低氧性肺动高压（HPH）的特征性病理变化，其确切机制不明。课题组实验表明：10%O2仅引起肺血管平滑肌细胞(VSMCs)增殖；3%O2可引起体、肺VSMCs增殖；但1%O2则抑制二者的增殖，提示体、肺VSMCs对低氧增殖的敏感性不同。我们提出：体、肺VSMCs存在不同的“低氧增殖反应阈”，试图解释肺泡低氧只发生肺动脉高压和肺VSMCs增殖的机制。鉴于ROS、HIF-1α及Skp2在低氧和VSAMCs增殖中关键作用，本研究项目拟从整体、细胞和分子水平观测不同浓度低氧对体、肺循环以及VSMCs增殖、凋亡的影响，确定体、肺VSMCs低氧增殖反应阈值，研究ROS，HIF-1α和Skp2/ P27通路在SMCs低氧阈形成中的作用及机制，为HPH和VSMCs增殖的发生机制提供新理论、为其防治提供新靶点。

课题组在以往的实验结果基础上提出：体、肺VSMCs存在不同的“低氧增殖反应阈”假说，试图解释肺泡低氧只发生肺动脉高压和肺动脉平滑肌细胞（PASMCs）增殖的机制。体外细胞培养实验显示， PASMCs在3%O2～12%O2浓度培养24 h出现明显的增殖反应，而主动脉平滑肌细胞（AASMCs）只有在3%O2浓度培养24h才出现低氧增殖反应，说明体、肺VSMCs对低氧的增殖反应阈存在差异。常氧条件下PASMCs与AASMCs存在857条差异表达基因、386个差异表达蛋白、237种差异代谢产物，在低氧时这些差异虽有变化，但差异依然存在。说明体、肺血管存在生物学差异，对低氧的反应也有区别。低氧（5%O2）可诱导PASMCs线粒体生产ROS，但不引起AASMCs的ROS增多；外源性H2O2可诱导VSMCs增殖，但体、肺VSMCs对H2O2的敏感性不同，清除ROS能够抑制VSMCs增殖。体内、外实验研究表明低氧可以下调PASMCs中PHD3的表达，引起PASMCs内HIF-1a明显增加，促进PDK2的表达,但不增加体循环的颈动脉平滑肌细胞（CASMCs）和肠系膜动脉平滑肌细胞（MASMCs）内的PHD3、HIF-1a和PDK2水平。抑制HIF-1a可抑制低氧性PASMCs增殖，表明HIF-1a是低氧性VSMCs增殖阈形成的关键因素之一。整体实验结果显示：低氧引起大鼠肺动脉压增高，同时肺血管壁中膜厚度以及右心室指数均增加，但对体循环压，颈动脉壁、肠系膜动脉壁厚度没有明显影响。上述结果从整体水平说明了体、肺循环血管对低氧的反应差异是内在因素所为，并非重力学因素所致。本课题组联合交通大学医学部药化研究组，合成并筛选出来了L1、L2和W922三种新合成的PI3K抑制剂，进一步研究结果显示，W922可以显著降低HPH大鼠的右室平均压，对肺血管壁增厚有抑制作用并呈剂量依赖性。该研究从细胞、动物和分子水平，观察了体、肺循环血管的生物学本质差异，明确了二者对于低氧反应的差异，对于正确认识HPH的发生发展具有重要的理论价值，对于进一步研发特异性靶向药物具有良好的启迪和指导作用。