FETAL LUNG BILAYER-- GLUTATHIONE TRANSPORT AND HYPEROXIA

肺不成熟会导致肺顺应性下降和呼吸窘迫。管理的支柱是氧气和呼吸支持。然而，这些疗法可能会导致氧化损伤，并导致进一步的肺部疾病。在氧化治疗中，抗氧化剂系统在减轻有毒氧中间体的负担和防止组织损伤方面起着至关重要的作用。在早产儿中，氧疗过程中产生的活性氧物质造成的损伤，加上不发达的抗氧化系统，使之更加复杂。谷胱甘肽(GSH)是肺部抗氧化系统的重要组成部分，早产与肺组织和肺泡表面液体中GSH减少有关。上皮衬膜液中GSH含量的降低及损伤程度与胎龄呈负相关，提示上皮衬膜液中GSH含量的重要性。上皮内膜液GSH的减少可能是由于血浆GSH的可获得性、微血管内皮细胞(MVEC)的摄取、肺泡上皮细胞(AEC)的摄取以及GSH在肺泡表面的释放所致。由于肺中有40多种不同的细胞类型，用体内和整个器官模型研究谷胱甘肽的转运很难解释。对培养的胎鼠MVEC或培养的AEC的研究将为GSH转运的发展以及高氧暴露对该转运的影响提供重要的见解。然而，对任何一种细胞类型的孤立研究只能提供部分见解。如果MVED和AEC都以适当的方向存在，则可以得到GSH转运的更完整的图像。利用CoStar Transwell，我们利用成年豚鼠的原代MVEC和AEC建立了一个双层模型，该模型的配置是MVEC基侧表面与AEC基侧表面相邻。AEC表面是空气界面而不是液体界面。该方案的目标是从不同胎龄的豚鼠体内分离出原代MVEC和AEC的胎儿双层(目标1)。这一胎儿双层随后将用于研究GSH从内皮表面到上皮表面的妊娠发育(目标2)以及高氧对该运输过程的影响(目标3)。这种胎儿双层的发育将是研究许多需要内皮和上皮细胞相互作用的生理过程的有用模型，包括中性粒细胞的黏附和迁移。

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.