剪接调控蛋白ESRP和NADPH氧化酶对人气道上皮间质转化的作用及机制研究

Small airway remodeling is the main contributor in airflow obstruction in COPD. Epithelial-mesenchymal transition(EMT) play an important role in this process. Quite recently, alternative splicing (AS) of pre-mRNAs was a novel regulation of EMT have been found and ESRP were suggested to be epithelial-specific AS factor which regulated EMT, by mechanisms that are far less well understood. Recent studies and data from our group suggest that TGFβ1 triggered intracellular ROS release by upregulation of NADPH oxidase 4 through Smad signaling and triggered deposition of ECM. In addition, our group also found recently that TGFβ1 induced NOX4 and ESRP expression in airway epithelial cells and accompany with EMT process. Together with the most recent results, prompt the hypothesis that NADPH modulated ROS production might induce EMT by interacting with EMT transcription factors and ESRP expression. The crosstalk between NOX4-ROS and ESRP mediated AS might involved in EMT process. The current project is aimed to explore the interactions of NADPH and ESRP and the signal of ESRP-mediated AS in EMT process. The impact of NADPH and ESRP on EMT in small airway is to be investigated in patients with COPD, animal model and in vitro cell cultures. The data from these studies will help to develop new therapeutic targets for small airway remodeling in COPD.

气道上皮细胞间质转化（EMT）是引起COPD气道重塑的重要环节。新近研究发现上皮细胞剪接调控蛋白（ESRP）介导的选择性剪接是EMT的重要调控机制，但其机制尚不明确。近期研究和我们前期的工作显示：TGFβ1可以通过活化smad信号通路诱导细胞内NOX4，产生内源性ROS，促进细胞外基质的合成。此外，我们新近发现：TGFβ1能诱导人气道上皮细胞NOX4和ESRP的高表达，促进上皮细胞向间质表型的转分化（EMT）。我们推测：NDAPH氧化酶-ROS信号通路可能通过与EMT转录调控因子相互作用和造成细胞内源性氧化应激的方式影响细胞内ESRP的表达，进而通过剪接调控的方式调控EMT的发生、发展。本课题将重点通过细胞、动物、人体三个层面的研究，探索NADPH氧化酶与ESRP剪接调控间的相互作用，论证ESRP剪接调控在上皮细胞间质转化的信号机制，以期探寻COPD气道重塑的新机制和药物治疗靶点。

气道上皮细胞间质转化（EMT）是引起COPD气道重塑的重要环节。我们前期的工作显示：TGFβ1可以通过活化smad信号通路诱导细胞内NOX4，产生内源性ROS，促进细胞外基质的合成是慢阻肺气道重塑的重要机制之一，此外，我们前期还发现TGFβ1能诱导剪接调控蛋白（ESRP）的活化及其下游EMT的发生发展，具体机制不明。因此，通过此项目研究我们深入探究了：1、基于慢阻肺患者临床标本的研究证实COPD患者存在明显的氧化应激，肺组织小气道存在EMT现象，且NOX4和ESRP1可能通过某种信号机制参与EMT的发生发展；2、细胞水平的研究结果证实：TGFβ1—NOX4—ROS信号通路在人气道上皮细胞EMT的发生发展中发挥重要作用，进一步研究证实ESRP1与NOX4之间存在相互交联，ESRP1通过参与NOX4的信号通路进而调控EMT的发生发展。抗氧化剂NAC能明显拮抗TGFβ1诱导的EMT的发生，NAC可作为COPD防治新的药物靶点。3、动物实验研究结果证实：COPD小鼠模型存在上皮细胞间质转化和平滑肌细胞增生为主要特点的气道重塑；烟熏小鼠肺组织中NOX4和ESRP1的表达增高、细胞外基质明显增生。ESRP及NOX4的表达与EMT呈正相关。基于过细胞、动物、人体三个层面的研究证实：COPD肺组织存在以上皮间质转化为主的EMT，TGFβ1—ESRP1-NOX4—ROS信号机制参与EMT的发生发展；抗氧化剂NAC能明显拮抗TGFβ1诱导的EMT，NAC可作为COPD防治新的药物靶点。