FETAL LUNG BILAYER-- GLUTATHIONE TRANSPORT AND HYPEROXIA

肺不成熟导致肺顺应性降低和呼吸窘迫。 管理的主要是氧气和呼吸支持。 然而，这些疗法可能导致氧化损伤并导致进一步的肺部疾病。 在氧化剂治疗过程中，抗氧化系统在减少有毒氧中间体的负担和防止组织损伤方面至关重要。 在早产儿中，氧疗过程中产生的活性氧损伤与发育不良的抗氧化系统有关。 谷胱甘肽（GSH）是肺抗氧化系统的重要组成部分，早产与肺组织和肺泡表面衬里液体中的GSH减少有关。 上皮衬里液中GSH含量的减少和损伤与胎龄呈负相关，提示上皮衬里液中GSH含量的重要性。 上皮衬里液GSH的这种减少可能是由于血浆GSH的可用性、微血管内皮细胞（MVEC）的摄取、肺泡上皮细胞（AEC）的摄取以及GSH释放到肺泡表面。 由于肺中有超过40种不同的细胞类型，因此难以解释体内和整个器官模型的GSH转运研究。 研究培养的MVEC或培养的AEC从胎儿动物将提供重要的洞察GSH运输的发展和高氧暴露对运输的影响。 然而，对任一细胞类型的孤立研究只能提供部分见解。 一个更完整的GSH运输的图片将获得一个模型，其中MVED和AEC都存在于适当的方向。使用CoStar Transwell，我们已经开发了一个双层模型，使用来自成年豚鼠的原代MVEC和AEC，其处于MVEC基底外侧表面与AEC基底外侧表面相邻的配置中。 AEC表面是空气界面而不是液体界面。 本提案的目标是开发具有从不同胎龄豚鼠中分离的原代MVEC和AEC的胎儿双层（目标1）。 然后将使用该胎儿双层研究GSH从内皮表面转运至上皮表面的妊娠发育（目的2）以及高氧对该转运过程的影响（目的3）。 这种胎儿双层的发展将是一个有用的模型，研究许多生理过程，需要内皮细胞和上皮细胞的相互作用，包括中性粒细胞的粘附和迁移。

Lung immaturity leads to decreased pulmonary compliance and respiratory distress. The mainstay in management is oxygen and respiratory support. However, these therapies can cause oxidative injury and lead to further pulmonary disease. During oxidant therapy, antioxidant systems are critical in reducing the burden of toxic oxygen intermediates and preventing tissue injury. In premature infants, injury from reactive oxygen species generated during oxygen therapy is compounded by a poorly developed antioxidant system. Glutathione (GSH) is an essential component of the pulmonary antioxidant system and premature birth is associated with decreased GSH in lung tissue and in the fluid lining the alveolar surface. The decrease in GSH of the epithelial lining fluid and the injury were inversely related to the gestational age and suggest the importance of the GSH content of the epithelial lining fluid. This decrease in the epithelial lining fluid GSH could be due to plasma GSH availability, uptake by microvascular endothelial cells (MVEC), uptake by alveolar epithelial cells (AEC) and release of GSH onto the alveolar surface. With over 40 different cell types in the lung, studies of GSH transport with in vivo and whole organ models are difficult to interpret. Studies of cultured MVEC or cultured AEC from fetal animals will provide important insight into the development of GSH transport and the impact of hyperoxic exposure on that transport. However, studies of either cell type in isolation will provide only partial insight. A more complete picture of GSH transport would be obtained with a model where both MVED and AEC were present in the appropriate orientation. Using a CoStar Transwell, we have developed a bilayer model using primary MVEC and AEC from adult guinea pigs that are in a configuration where the MVEC basolateral surface in adjacent to the AEC basolateral surface. The AEC surface is an air interface rather than a liquid interface. The goal of this proposal is to develop a fetal bilayer with primary MVEC and AEC isolated from guinea pigs at different gestational ages (Aim 1). This fetal bilayer will then be used study gestational development of GSH transport from the endothelial surface to the epithelial surface (Aim 2) and the impact of hyperoxia on that transport process (Aim 3). The development of this fetal bilayer will be a useful model to study many physiological processes that require endothelial and epithelial cell interaction including neutrophil adhesion and migration.