Pathogenesis of Airway Stem Cell Abnormalities in Obliterative Bronchiolitis

项目摘要： 终末期肺病是世界范围内发病率和死亡率的主要原因。肺移植是一种 对于患有这种疾病的患者来说，这是一种极好的治疗选择，但50%的接受者在五年内死亡， 在同种异体移植物中发生闭塞性细支气管炎（OB）。建议去除上皮干细胞， 有助于OB的发展;然而，很少有研究来检验这一假设， 这主要是由于缺乏OB的动物模型，这些动物模型发生类似于在 人类我们最近开发了一种新的原位肺移植模型，在雪貂模型人类OB 很好.利用这个模型，我们首次在雪貂和人类同种异体移植物中发现， 克隆原性K5+p63+基底干细胞（BSC）在同种异体移植物的近端和远端气道中进行性下降 随着OB严重程度的增加。此外，我们的研究首次证明近端气道 粘膜下腺（SMG）是支气管上皮细胞（SAE）的兼性小生境，是早期靶点 在人类和雪貂同种异体移植气道中的免疫破坏。在小鼠中，SMG干细胞龛仅服务于 然而，在较大的哺乳动物，如人类和雪貂，SMG存在于整个气管， 软骨气道使用谱系追踪，我们已经表明SMG的肌上皮细胞（MEC）是 SAE中多能K5+p63+ BSC的前体。在OB的发展过程中，SMG的破坏 同种异体移植物中的干细胞龛与表型和功能变化同时发生， SAE中的K5+p63+ BSC。我们假设同种异体移植物中失神经SMG的破坏， MEC的耗竭导致SAE中多能性K5+p63+ BSC的减少，并导致严重急性胰腺炎中定向K5+p63+ BSCs的增加。 多能（K5+p63+K14+）、双能（K5+K14+，p63+K14+）和单能（K14+）基底细胞，所有这些细胞都具有 自我更新能力下降。本研究的目的是确定 MECS和谱系定向基底细胞的表型变化的意义以及去神经支配如何 SMG改变了维持SMG干细胞生态位所需的Wnt信号（Lef-1/TCF 1）， 在损伤的情况下，腺MEC向SAE BSC的谱系定型。此外，一个重要的临床前 这项计划的目的是阐明干细胞植入移植肺并修复的能力。 从而为发展干细胞治疗延缓或预防肺内OB奠定了基础 同种异体移植我们将通过以下具体目标实现这些目标：1）确定如何 SMG干细胞小生境的破坏有助于K5+p63+ BSC的耗尽。2)确定以下后果 肺去神经对气道干细胞及其小生境的影响。3)确定气道干细胞的潜力 移植预防或延缓OB。我们希望阐明干细胞耗竭的机制 在移植肺的气道中，将是理解OB发病机制的重要一步， 告知使用干细胞作为预防或延迟OB的治疗方法。

Project Summary: End-stage lung diseases are a major cause of morbidity and mortality worldwide. Lung transplantation is an excellent treatment option for patients with this condition, yet 50% of recipients die within five years due to the development of obliterative bronchiolitis (OB) in the allograft. Epithelial stem cell depletion is suggested to contribute to the development of OB; however, there is little research being performed to test this hypothesis, primarily due to the lack of animal models for OB that develop allograft pathology resembling that seen in humans. We recently developed a novel orthotopic lung transplant model in the ferret that models human OB very well. Using this model, we have shown for the first time in ferret and human allografts that the number of clonogenic K5+p63+ basal stem cells (BSCs) progressively declines in proximal and distal airways of the allograft as the severity of OB increases. Additionally, our research is the first to demonstrate that the proximal airway submucosal gland (SMG), a facultative niche for BSCs in the surface airway epithelium (SAE), is an early target of immune destruction in human and ferret allograft airways. In mice, the SMG stem cell niche serves only the trachea; however, in larger mammals such as humans and ferrets, SMGs are present throughout the cartilaginous airways. Using lineage tracing, we have shown that the myoepithelial cells (MECs) of SMGs are precursors of multipotent K5+p63+ BSCs in the SAE. During the development of OB, the destruction of SMG stem cell niches in the allograft occurs simultaneously with phenotypic and functional changes to multipotent K5+p63+ BSCs in the SAE. We hypothesize that destruction of the denervated SMG in the allograft, and thus depletion of MECs, leads to a decline in multipotent K5+p63+ BSCs in the SAE, and to increases in committed multipotent (K5+p63+K14+), bipotent (K5+K14+, p63+K14+) and unipotent (K14+) basal cells, all of which have a reduced capacity for self-renewal. The objective of the proposed research is to determine the functional significance of the phenotypic changes in the MECS and the lineage-committed basal cells and how denervation of the SMG alters Wnt signaling (Lef-1/TCF1) required for both the maintenance of the SMG stem cell niche and lineage commitment of glandular MECs to SAE BSCs in the setting of injury. Additionally, a major preclinical objective of this proposal is to elucidate the ability of stem cells to engraft into a transplanted lung and repair injury, thus laying the foundation for the development of stem cell therapy to delay or prevent OB in lung allografts. We will achieve these objectives by addressing the following specific aims: 1) Determine how destruction of the SMG stem cell niche contributes to depletion of K5+p63+ BSCs. 2) Identify consequences of lung denervation on airway stem cells and their niches. 3) Determine the potential for airway stem cell transplantation in preventing or delaying OB. We expect that delineating the mechanisms of stem cell depletion in the airways of transplanted lungs will be a major step forward in understanding OB pathogenesis and will inform the use of stem cells as a therapeutic approach to prevent or delay OB.