The role of resident lung mesenchymal stromal cells in the pathogenesis of bronchopulmonary dysplasia (BPD).

支气管肺发育不良（BPD）是急性呼吸衰竭后使用呼吸机和氧气治疗的慢性肺部疾病，是早产最常见的并发症，也是长期发病率的主要原因。BPD的特点是肺部发育受损，缺乏有效的治疗方法。外源性（骨髓或脐带）间充质间质细胞（MSC）可预防新生啮齿动物o2诱导的肺损伤。一个明显的矛盾是观察到，如果气管吸入物中存在间充质干细胞，早产儿更有可能发展成BPD。因此，为了促进这种有前景的细胞疗法的临床转化，我们将阐明常驻肺（L-）间充质干细胞在新生儿肺损伤和修复中的有趣作用。假设：实验性BPD中L- msc的修复功能受到干扰，L- msc功能障碍可能与BPD的发病机制有关。目的1：表征正常发育小鼠肺中L-MSCs的特征，确定实验性BPD中L-MSCs的功能是否受到干扰。1.1. 我们将从正常小鼠中分离出L-MSCs，并确定其特征（细胞表面标记物、分化和克隆潜能、细胞因子和细胞外基质蛋白的产生）。1.2. 我们将使用相同的终点来比较正常小鼠和模拟BPD的通气性肺损伤（VILI）小鼠的L-MSCs。小鼠将暴露于LPS并机械通气8小时。我们还将使用RNAseq来比较正常L-MSC与VILI小鼠分离的L-MSC的转录。此外，我们将进行体外功能测定来比较L-MSCs的愈合潜力。我们将比较来自对照小鼠和VILI小鼠的L-MSCs对新分离肺泡上皮细胞增殖和伤口愈合以及肺内皮细胞基质增殖和网络形成的影响。目的2：确定L-MSCs是否参与BPD的发病机制。在体内，我们将在P4（肺泡发育开始）时向正常小鼠幼崽或暴露于高氧环境（n=10/组）的幼崽注射对照L-MSCs（先前从正常发育小鼠中分离的）或从VILI小鼠中分离的L-MSCs，并比较P14时对肺功能和结构的影响。该建议的优势包括探索内源性L-MSCs作用的新颖性，易于获得的动物模型，成熟的分离和表征L-MSCs的技术，肺功能和结构以及直接的临床意义。事实上，体外功能测定可以作为效力测定来预测细胞的体内生物活性。此外，更好地了解L-MSCs的组织特异性功能可能有助于开发更好的外源性mscs为基础的疗法。我们的发现的相关性可能扩展到以肺泡损伤为特征的成人肺部疾病，如肺气肿和急性呼吸窘迫综合征。

Bronchopulmonary dysplasia (BPD), the chronic lung disease that follows ventilator and O2 therapy for acute respiratory failure, is the most common complication of prematurity and accounts for much of the long-term morbidity. BPD is characterized by impaired lung development and lacks effective therapies. Exogenous (bone marrow or umbilical cord) mesenchymal stromal cells (MSC) prevent O2-induced lung injury in newborn rodents. An apparent contradiction is the observation that premature babies are more likely to develop BPD if MSCs are present in their tracheal aspirates. Thus, in order to facilitate the clinical translation of this promising cell therapy, we will clarify the intriguing role of resident lung (L-)MSC in neonatal lung injury and repair.Hypothesis: The repair function of L MSCs is perturbed in experimental BPD and L-MSC dysfunction may contribute to the pathogenesis of BPD. Aim 1: To characterize L-MSCs in the normal developing mouse lung and determine if the function of L-MSCs is perturbed in experimental BPD. 1.1. We will isolate L-MSCs from normal mice and determine their characteristics (cell surface markers, differentiation and clonogenic potential and cytokine and extracellular matrix protein production). 1.2. We will use these same endpoints to compare L-MSCs from normal mice to mice with ventilation-induced lung injury (VILI) mimicking BPD. Mice will be exposed to LPS and mechanically ventilated for 8 hours. We will also perform RNAseq to compare the transcription of normal L-MSC compared to L-MSC isolated from mice with VILI. In addition, we will perform in vitro functional assays to compare the healing potential of L-MSCs. We will compare the effect of L-MSCs from control mice and from VILI mice on proliferation and wound healing of freshly isolated alveolar epithelial cells and proliferation and network formation on matrigel of lung endothelial cells.Aim 2: To determine if L-MSCs contribute to the pathogenesis of BPD. In vivo, we will inject normal mouse pups or pups exposed to hyperoxia (n=10/group) at P4 (onset of alveolar development) with either control L-MSCs (isolated previously from normally developing mice) or L-MSCs isolated from VILI mice and compare the effect on lung function and structure at P14. The strengths of this proposal include the novelty in exploring the role of endogenous L-MSCs, the readily availability of the animal models, well-established techniques to isolate and characterize L-MSCs, lung function and structure as well as the direct clinical implications. Indeed, the functional in vitro assays may serve as potency assays to predict the in vivo bioactivity of the cells. Furthermore, a better understanding of L-MSCs tissue specific function may contribute to develop superior exogenous MSC-based therapies. The relevance of our findings may extend to adult lung diseases characterized by alveolar damage such as emphysema and acute respiratory distress syndrome.