Fang Xiaohui, Song Yuanlin, Zemans Rachel, Hirsch Jan, Matthay Michael A
Cardiovascular Research Institute, University of California, San Francisco, 94143-0130, USA.
Am J Physiol Lung Cell Mol Physiol. 2004 Jul;287(1):L104-10. doi: 10.1152/ajplung.00176.2003. Epub 2004 Feb 27.
Previous studies have used fluid-instilled lungs to measure net alveolar fluid transport in intact animal and human lungs. However, intact lung studies have two limitations: the contribution of different distal lung epithelial cells cannot be studied separately, and the surface area for fluid absorption can only be approximated. Therefore, we developed a method to measure net vectorial fluid transport in cultured rat alveolar type II cells using an air-liquid interface. The cells were seeded on 0.4-microm microporous inserts in a Transwell system. At 96 h, the transmembrane electrical resistance reached a peak level (1,530 +/- 115 Omega.cm(2)) with morphological evidence of tight junctions. We measured net fluid transport by placing 150 microl of culture medium containing 0.5 microCi of (131)I-albumin on the apical side of the polarized cells. Protein permeability across the cell monolayer, as measured by labeled albumin, was 1.17 +/- 0.34% over 24 h. The change in concentration of (131)I-albumin in the apical fluid was used to determine the net fluid transported across the monolayer over 12 and 24 h. The net basal fluid transport was 0.84 microl.cm(-2).h(-1). cAMP stimulation with forskolin and IBMX increased fluid transport by 96%. Amiloride inhibited both the basal and stimulated fluid transport. Ouabain inhibited basal fluid transport by 93%. The cultured cells retained alveolar type II-like features based on morphologic studies, including ultrastructural imaging. In conclusion, this novel in vitro system can be used to measure net vectorial fluid transport across cultured, polarized alveolar epithelial cells.
以往的研究曾使用灌注液体的肺来测量完整动物和人类肺中的肺泡液体净转运。然而，完整肺研究存在两个局限性：不同远端肺上皮细胞的贡献无法单独研究，并且液体吸收的表面积只能大致估算。因此，我们开发了一种方法，利用气液界面来测量培养的大鼠II型肺泡细胞中的液体净矢量转运。将细胞接种在Transwell系统中的0.4微米微孔插入物上。在96小时时，跨膜电阻达到峰值水平（1530±115Ω·cm²），并有紧密连接的形态学证据。我们通过在极化细胞的顶端侧放置150微升含有0.5微居里¹³¹I - 白蛋白的培养基来测量液体净转运。通过标记白蛋白测量的跨细胞单层的蛋白质通透性在24小时内为1.17±0.34%。顶端液体中¹³¹I - 白蛋白浓度的变化用于确定在12小时和24小时内跨单层转运的液体净量。基底液体净转运为0.84微升·厘米⁻²·小时⁻¹。用福斯可林和异丁基甲基黄嘌呤刺激cAMP可使液体转运增加96%。氨氯吡咪抑制基础和刺激后的液体转运。哇巴因抑制基础液体转运93%。基于形态学研究，包括超微结构成像，培养的细胞保留了II型肺泡样特征。总之，这种新型体外系统可用于测量跨培养的、极化的肺泡上皮细胞的液体净矢量转运。
