Pathophysiological studies in neonatal tracheal aspirate derived mesenchymal stromal cells and animal models to determine the role of Pbx1 and other candidate genes in congenital diaphragmatic hernia.

我们提出这一建议的目的是通过细胞和动物模型来了解转录因子Pbx1在先天性横隔疝(CDH)的病理生理学中的作用。CDH的特征是在胚胎发育过程中发生的横隔膜缺陷，并与肺发育不全和持续性肺动脉高压有关。受影响新生儿的高死亡率(30%-50%)在很大程度上取决于肺发育不良的程度，其潜在的病理生物学尚不清楚。我们从全基因组关联研究得知，转录因子Pbx1是CDH的候选基因，在小鼠中敲除Pbx1会导致肺发育不良和高血压，就像在CDH患者中看到的那样。Pbx1的表达一直持续到出生后，直接刺激Pbx1下游靶点可以挽救受损肺的发育。因此，它作为产后干预的治疗靶点具有潜在的意义。在这里，我们的目的是描述Pbx1在CDH所致的正常和异常肺发育中的作用。我们将使用从CDH和“肺健康”对照新生儿气管抽吸物中分离的间充质基质细胞，并进行不同的机制研究。我们将利用CRISPR/Cas9介导的Pbx1在这些间充质基质细胞中的敲除，并测试这如何影响细胞形态，并确定这些细胞的表观遗传特征的变化。此外，我们还将获得不同发育时间点各自细胞的蛋白质组学图谱，以分析蛋白质水平的变化。当它们出现时，其他候选基因将与已建立的工作管道进行相应的测试。最后，CDH气管抽吸物细胞将接受基因替换或药物治疗，这些药物旨在调节或扰乱蛋白质相互作用分析确定的通路。

Our goal with this proposal is to understand the role of the transcription factor Pbx1 in the pathophysiology of congenital diaphragmatic hernia (CDH) using cell-based and animal models. CDH is characterized by a diaphragmatic defect that occurs during embryonic development and is associated with pulmonary hypoplasia and persistent pulmonary hypertension. The high mortality (30-50%) of affected neonates depends largely on the degree of pulmonary hypoplasia, of which the underlying pathobiology is not sufficiently understood. We know from genome wide association studies, that transcription factor Pbx1 is a candidate gene in CDH and knockdown of Pbx1 in mice results in pulmonary hypoplasia and hypertension as seen in CDH patients. Pbx1 is expressed until the postnatal period and direct stimulation of Pbx1 downstream targets can rescue compromised lung development in affected lungs. Therefore, it is potentially relevant as a therapeutic target for postnatal interventions. Here, we aim to describe the role of Pbx1 in normal and abnormal lung development due to CDH. We will use mesenchymal stromal cells isolated from tracheal aspirates from intubated neonates with CDH and “lung healthy” controls and perform different mechanistic studies. We will leverage CRISPR/Cas9 mediated knockdown of Pbx1 in these mesenchymal stromal cells and test how this affects cell morphology and determine changes in epigenetic signature of these cells. Moreover, we will obtain proteomic profiles of the respective cells at different developmental timepoints to analyze the changes on protein level. As they emerge other candidate genes will be tested accordingly with the established work pipeline. Finally, CDH tracheal aspirate cells will be subjected to gene replacement or treatment with drugs designed to regulate or perturb pathways identified by protein interaction analyses.