HSP70调控HIF-1α在缺氧性肺动脉高压新生大鼠肺血管内皮细胞凋亡中的作用机制研究

Neonatal hypoxic pulmonary hypertension (HPH) is one of the serious diseases leading to death in the neonates, which need early intervention for the vasospasm change in the early stage is reversible, while the vascular remodeling in the late stage is difficult to treat. In our previous study, we found that Heat Shock Protein 70(HSP70) can down-regulate the expression of HIF-1α and its downstream target gene, and therefore deduce pulmonary artery pressure.Hypoxia can induce apoptosis of pulmonary vascular endothelial cell, vasospasm, and was closely related with its early onset of HPH, which were based on the studies of tumors and HPH study in adults. BAG3/HSP70/Bax signaling pathway may be involved in the regulation of HIF-1α up-regulation, which may regulated the cell apoptosis and be related to the pathogenesis of PAH. The pathogenesis of HPH in neonatal HPH is unclear. The aim of this study is to investigate the relationship between the pulmonary artery pressure pulmonary, vascular endothelial cell apoptosis and vascular spasm in the newborn rat with HPH, and whether HSP70 down-regulates the expression of HIF-1α, through in vitro and in vivo experiments by Laser capture microdissection (LCM), RNA interference (RNAi) and co-immunoprecipitation (CO-IP), through reducing the pulmonary vascular endothelial cell apoptosis and reducing pulmonary artery pressure in neonatal rats. To further study from the beginning of the link to find accurate prevention and treatment targets, to provide a basis for blocking its course accurately.

新生儿缺氧性肺动脉高压（HPH）是导致其死亡的严重疾病之一，早期肺血管痉挛可逆，晚期肺血管重塑救治困难，发病机制不清，治疗受限。本课题组前期研究发现热休克蛋白70（HSP70）能够下调缺氧诱导因子-1α(HIF-1α)及其下游靶基因表达，降低肺动脉压力。成人肿瘤及HPH研究发现缺氧可诱导肺血管内皮细胞凋亡，血管痉挛，与早期发病密切相关；HSP70可以通过BAG3/HSP70/Bax信号通路参与HIF-1α上游调控及细胞凋亡而参与其发病机制。但在新生儿HPH发病机制中是否起同样作用仍不清楚。本课题在前期研究基础上拟从体内、外实验通过激光捕获纤维切割、RNA干扰、免疫共沉淀等技术探讨HPH新生大鼠肺血管内皮细胞凋亡和血管痉挛、肺动脉压力的关系，以及HSP70是否通过此通路下调HIF-1α表达，减轻血管内皮细胞凋亡，降低肺动脉压力。为进一步研究从始动环节寻找早期精准防治靶点提供依据。

新生儿HPH具体发病机制不清，本课题组查找相关文献发现肺血管内皮细胞（PVECs）凋亡是成人HPH发病的起始环节，缺氧状态下HIF-1α大量表达能够诱导该细胞凋亡，可能促进该病的发生及发展。课题组前期研究表明HSP70通过下调HIF-1α发挥了保护HPH新生大鼠肺血管的作用，另外，成人肿瘤研究发现HSP70通过BAG3/HSP70/Bax信号通路下调HIF-1α表达从而抑制了肿瘤细胞凋亡，而在新生儿HPH发病过程中HSP70是否通过该信号通路调控HIF-1α表达从而抑制了PVECs凋亡，进而发挥了延缓或阻断该病进程的作用，需要进一步深入研究。本研究通过对新生大鼠PVECs进行缺氧、HSP70慢病毒和BAG3 siRNA质粒转染以及HSP70抑制剂KNK437等干预处理后，借助免疫共沉淀、流式细胞术、RT-qPCR、Western blotting等方法发现新生大鼠PVECs在缺氧早期以线粒体途径的凋亡表型为主，抑凋亡因子HSP70通过BAG3/HSP70/Bax信号通路抑制了HIF-1α表达从而减轻了PVECs线粒体途径的凋亡；本研究在前期建立新生大鼠HPH模型基础上，将HSP70过表达腺病毒经气管内途径成功转染至新生大鼠肺组织，利用小干扰RNA、免疫组化、免疫双标、电镜技术等实验方法发现PVECs凋亡是新生大鼠HPH病变的早期环节，该细胞凋亡促进了肺动脉压力升高及肺血管重塑，HSP70可能通过BAG3/HSP70/Bax信号通路下调HIF-1α表达抑制了PVECs凋亡从而降低了HPH新生大鼠肺动脉压力及肺血管重塑程度，以上研究结果为寻找延缓甚至阻断新生儿HPH疾病进程的新靶点提供了基础理论依据。