Mangum J B, Everitt J I, Bonner J C, Moore L R, Brody A R
Department of Experimental Pathology and Toxicology, CIIT, Research Triangle Park, North Carolina.
In Vitro Cell Dev Biol. 1990 Dec;26(12):1135-43. doi: 10.1007/BF02623690.
Primary rat alveolar type II cells and early passage rat lung fibroblasts were co-cultured on opposite sides of a collagen-coated polycarbonate filter. This is an approach to "model", in part, an alveolar wall to study mechanisms of cytotoxicity and translocation of bioactive materials from the alveolar space to the lung interstitium. Type II cells were recovered from adult rat (Fischer 344) lungs by enzyme digestion and "panning". Lung fibroblasts were separated from the same species, cultured initially in 10% fetal bovine serum and used in the co-culture system at early passage. The type II cells formed a monolayer of dedifferentiated epithelium which provided a barrier on the upper side of the collagen (human type IV)-coated filter. The fibroblasts on the bottom of the filter replicated logarithmically in the presence of serum, could be rendered quiescent in defined medium and then returned to rapid growth phase with the reintroduction of serum. The intact epithelial monolayer excluded trypan blue, albumin, platelet-derived growth factor, and alpha2-macroglobulin from the lower compartment of the culture chamber. Altering the integrity of the monolayer by a variety of means allowed translocation of these materials through the collagen-coated filters. Particularly interesting was the effect of taurine chloramine which caused subtle changes in the alveolar epithelium and allowed subsequent translocation of albumin. In addition, we showed that rat alveolar macrophages remain viable with some spreading on the surface of the epithelial monolayer. This co-culture system will have future application in the study of how reactive oxygen species might affect the epithelial barrier, and whether macrophage-derived growth factors can influence fibroblast proliferation if the monolayer is intact or injured.
将原代大鼠肺泡II型细胞和早期传代的大鼠肺成纤维细胞在胶原包被的聚碳酸酯滤膜的相对两侧进行共培养。这是一种部分“模拟”肺泡壁的方法，用于研究细胞毒性机制以及生物活性物质从肺泡腔向肺间质的转运。II型细胞通过酶消化和“淘选”从成年大鼠（Fischer 344）肺中分离获得。肺成纤维细胞从同一物种分离，最初在含10%胎牛血清的培养基中培养，并在早期传代时用于共培养系统。II型细胞形成了一层去分化的上皮单层，在胶原（人IV型）包被的滤膜上侧形成了一道屏障。滤膜底部的成纤维细胞在有血清存在的情况下呈对数生长，在限定培养基中可进入静止期，重新加入血清后又可恢复快速生长阶段。完整的上皮单层可阻止台盼蓝、白蛋白、血小板衍生生长因子和α2-巨球蛋白进入培养室的下室。通过多种方式改变单层的完整性可使这些物质透过胶原包被的滤膜发生转运。特别有趣的是氯胺牛磺酸的作用，它会引起肺泡上皮的细微变化，并使白蛋白随后发生转运。此外，我们还表明大鼠肺泡巨噬细胞在这种上皮单层表面保持存活并会有一定程度的铺展。这种共培养系统在研究活性氧如何影响上皮屏障，以及如果单层完整或受损时巨噬细胞衍生的生长因子是否能影响成纤维细胞增殖方面将有未来应用。
