肺泡化阻滞的新机制：RhoA活性的时空变化介导TGF-α浓度梯度引起的肌成纤维细胞迁移的导向异常

Bronchopulmonary dysplasia (BPD) is characterized by alveolar developmental arrest with decreased saccular airway branching and fewer, larger alveoli. As it is clear that alveolar myofibroblasts are critical for the formation of secondary septa, their absence is associated with a lack of secondary septation and alveolarization failure. During alveolar development, alveolar myofibroblasts are found at the tips of secondary septa, and are associated with extracellular collagen and elastic fibers deposition. Thus, abnormal localization of myofibroblasts may result in abnormal elastin deposition, suggesting proposed functions of the alveolar myofibroblasts must take into account their topographical localization. We have demonstrated that intra-amniotic injection of LPS disrupted alveolar development and led to abnormal localization of myofibroblasts in the lung of newborn rats, mostly in primary septa with few in secondary septa. We found that LPS increased TGF-α expression and activated EGFR signaling both in vivo and vitro. LPS exposure, in vitro, disrupted the asymmetric localization of phospho-EGFR and the directional persistence during the myofibroblasts migration. Inhibition of EGFR signaling abrogated the enhanced locomotivity of myofibroblasts by LPS, and also increased the directional persistence of myofibroblasts migration. These results suggest the abnormal guidance of myofibroblasts migration may contribute to the abnormal location of myofibroblasts in vivo and further arrest lung development in BPD patients. However, it remains unclear how TGF-α influences the guidance of myofibroblasts migration. Each guidance molecule must activate a cascade of cytoplasmic effectors that eventually results in the cytoskeleton rearrangement. RhoA has emerged as key regulators of cytoskeletal rearrangements.We thus examined the involvement of RhoA in the regulation of myofibroblasts migration by TGF-α. We are to study whether TGF-α / EGFR signaling induces spatiotemporal dynamics of RhoA activity through DLC1 interacts with 14-3-3β protein and this appears to contribute to the abnormal of myofibroblasts migration. Furthermore, the lungs of α-SMA/EYFP mice were carried out to observe the migration behavior of myofibroblasts with time-lapse imaging. We also investigated whether the TGF-α is expressed in a gradient across the the lung induced by LPS, in which myofibroblasts were attractive or repulsive by TGF-α. The gradient of TGF-α may serve as a guidance signal for myofibroblasts migration, and then disrupt direction of cell migration, leading to alteration of the positioning of myofibroblast. These cells may not be in the correct location to promote alveolar septation and maturation of the alveolar, and may contribute to the arrested alveolarization in BPD. It is beneficial to understand the pathogenesis of BPD patients and lung development.

支气管肺发育不良（BPD）和肺泡发育阻滞有关，肌成纤维细胞（MF）在肺泡化过程中起关键作用。前期研究发现在羊膜腔注射LPS的BPD模型中肺MF分布异常；体外实验揭示LPS通过TGF-α/EGFR干扰MF的迁移方向。据此，我们提出了BPD肺泡化阻滞的新机制：肺发育过程中MF迁移的导向异常。本课题将探索TGF-α影响MF导向的途径，检测调节细胞骨架的中心环节-RhoA在TGF-α引发的迁移变化中的参与及信号传导，即TGF-α/EGFR是否通过DLC1结合14-3-3β，使RhoA活性发生时空的变化，致迁移方向发生改变。此外，用α-SMA/EYFP小鼠、细胞迁移实时观察等技术，证明在动物体内LPS诱导的肺组织中是否存在TGF-α浓度梯度，对MF迁移起吸引或排斥性导向作用，使MF不能按照原来的运动轨迹正确到达靶位点次级分隔顶端，从而阻断肺泡化过程。研究结果有助理解BPD发病机制及更清楚解读肺发育。

支气管肺发育不良（bronchopulmonary dysplasia, BPD）主要表现为肺泡数量减少，肺组织结构简单化。胎儿未成熟的肺暴露于宫内炎性环境中，是发生BPD 的主要危险因素。但是炎症BPD的相关机制并不是十分清楚，如何改善BPD也是近年来的热点之一。以下本研究的主要发现：.1.LPS抑制HDAC1和HDAC2的表达和活力，可引起TGF-α的表达增多，导致肺泡化阻滞。增强HDAC2表达和活力，可减弱TGF-α在BPD发生发展中的作用，而通过茶碱上调HDAC活性后，改善肺泡发育受损。.2.茶碱可抑制LPS引起的多种促炎因子的释放，同时促进抗炎因子的表达，其对促炎因子和抗炎因子平衡的调节作用可能是改善BPD肺泡发育受损的机制。.3.LPS通过下调Csk活性，激活Src，从而反式激活EGFR，影响RhoA活性，引起细胞定向迁移能力受损。Csk / Src / EGFR这条信号通路在调节肌成纤维细胞的定向迁移中有着至关重要的作用，激活该通路是LPS诱发BPD肺泡化阻滞的可能机制。.4.羊膜腔同时注射EGFR抑制剂Erlotinib可以改善LPS引起的新生鼠肺组织病理改变，提示LPS引起Csk，Src以及EGFR活性变化，导致肌成纤维细胞定向迁移能力受损可能是BPD病理机制中的重要环节.5. VitD通过下调IFN-γ和IL-1β的表达，改善LPS诱发BPD新生鼠的肺病理改变。