MiR-126促进BPD继发肺动脉高压EndMT分子机制研究

Despite advances in the care of extremely low birth weight infants, these infants remain at high risk of bronchopulmonary dysplasia (BPD). Pulmonary arterial hypertension (PAH) is an increasingly recognized complication of premature birth and BPD, and is associated with increased morbidity and mortality. Infants about 18% with BPD develop PAH. Since its original description in the late 1960's, the definition of BPD has changed dramatically from a description of stiff surfactant deficient lungs that were damaged by conventional ventilators to a more modern definition that describes lung growth arrest and decreased alveolarization. Consequently, understanding the pathophysiology of PAH in the BPD population remains unknown. Based on our original researches, we used C57BL/6 and miR-126-/- mice to establish BPD model and measured the pulmonary vascular-related protein expression at the 3, 7, 14, 21days after birth. We studied endothelial-mesenchymal transition (EndMT) of cultured pulmonary microvascular endothelial cells and the relationship between the miR-126, VEGFA/VEGFR2/PIK3R2 and VEGFA/VEGFR2/SPRED-1 signaling in cells and lung tissue levels. It may suggest that early intervention can reverse or reduce the incidence of EndMT in BPD associated PAH.

随着超低出生体重儿存活率的显著提高，支气管肺发育不良(Bronchopulmonary dysplasia,BPD)发生率亦呈逐年上升趋势，其中18%的BPD患儿将发展为不同程度的肺动脉高压(pulmonary arterial hypertension,PAH)。肺微血管异常发育在BPD患儿发生PAH中的作用日益受到关注但仍不明确。本研究分别采用C57BL/6和miR-126-/-小鼠建立BPD模型，研究生后3,7,14,21天的肺血管重建相关蛋白表达与内皮细胞间充质细胞转化(Endothelial-mesenchymal transition,EndMT)，分析VEGFA/VEGFR2/PIK3R2和VEGFA/VEGFR2/SPRED-1信号通路在单细胞和组织水平与miR-126之间的分子机制，进一步探讨早期抑制miR-126的合成是否能逆转或减轻BPD继发PAH的EndMT发生。

支气管肺发育不良（Bronchopulmonary dysplasia, BPD）是早产儿最常见的慢性肺部疾病，病死率高，存活者常遗留高反应性气道疾病、反复下呼吸道感染、生长发育迟缓。目前，早产儿、尤其是超产儿存活率明显提高，但BPD的发病率一直保持稳定或随着时间的推移而增加，胎龄小于28周的早产儿BPD的发生率仍在48～68%之间，极低出生体重儿在住院期间大约有25～40%被确诊为BPD，其中18%将发展为不同程度的肺动脉高压。BPD继发肺动脉高压死亡率高达14～38%。本研究采用C57BL/6小鼠建立BPD模型，研究生后3、7、14、21天肺血管的组织学特征，评估肺小动脉重建状态，研究Tek/p-ERK/ERK和IGF2/IGF1R/IGF2R信号通路在单细胞和组织水平与miR-34a-5p之间的关系，并将预处理miRCURY LNA miRNA Power Inhibitors小鼠或miR-34a-/-突变体小鼠建立BPD模型，探讨早期干预是否能减轻BPD EndMT的发生。研究结果发现，BPD的肺小动脉存在结构重建并且伴随肺泡简单化过程。为验证BPD间歇低氧下是否更容易造成肺小动脉的血管重建，免疫组化结果显示BPD组在生后第7、14、21天CD31表达降低，而Western blot结果在第7天无显著差异，但在第14天和第21天CD31的表达也有所下降。BPD组在第14天α-SMA明显升高，BPD间歇低氧后，α-SMA升高更为显著。同时，本研究通过miRNA-mRNA测序筛选和双荧光素酶报告实验验证Tek、IGF2的3’UTR能与miR-34a-5p结合，且结合位点为本实验验证位点。miR-34a-5p参与高氧诱导BPD肺血管EndMT与肺泡简化之间潜在的病理生理机制可能是通过Tek、IGF2进而介导p-ERK/ERK信号通路发挥作用。抑制miR-34a-5p表达，能减轻EndMT的发生，减轻肺泡简单化过程，以及Tek/p-ERK/ERK，IGF2/IGF1R/IGF2R信号通路有一定相关性。此外，高氧诱导BPD肺血管EndMT与肺泡简单化之间的潜在病理生理变化不仅仅涉及肺微血管内皮细胞的单一变化，此复杂过程亦涉及其他多种参与肺发育细胞之间的交互作用，在发病早期有效阻断某些关键转录因子的表达，可能会有效延缓发病进程。