Kinnard W V, Tuder R, Papst P, Fisher J H
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Am J Respir Cell Mol Biol. 1994 Oct;11(4):416-25. doi: 10.1165/ajrcmb.11.4.7917310.
Alveolar type II cells produce pulmonary surfactant and serve as the stem cell of the alveolar epithelium by proliferating and transforming into type I cells. The study of the differentiated function and proliferative capacity of type II cells in response to injury in vivo has been hindered by the complexity of the systemic response to injury. In vitro studies have in turn been limited by the impaired proliferative potential and loss of markers of differentiation in isolated type II cells maintained in culture. We describe an in vitro system in which type II cells proliferate spontaneously and simultaneously maintain differentiated characteristics. Other investigators have maintained slices of adult lung in culture after agarose infusion for up to 9 wk. To further develop this model for the study of epithelial cell differentiation and proliferation, we assessed epithelial differentiation, proliferative capacity, and regulation of cell-specific gene expression in slice explants of agarose-infused rat lungs. We prepared 1-mm-thick explants and maintained them in culture for up to 2 wk. Maintenance of differentiation was confirmed morphologically by light and electron microscopy, by the accumulation of epithelial cell-specific surfactant proteins, and by phospholipid analysis. Proliferative capacity was assessed by measuring [3H]thymidine incorporation in alveolar and small airway cells at baseline and in response to growth stimuli. Type II cell proliferation was inhibited in a dose-dependent manner by glucocorticoids. Glucocorticoids regulated RNA levels in explants in a manner similar to that seen in vivo and in fetal lung explants. The alveolar epithelium in adult lung slice explants maintains differentiated function and the ability to proliferate, thereby providing a useful system for the study of distal airway and alveolar cell homeostasis and response to injury.
II型肺泡细胞产生肺表面活性物质，并通过增殖和转化为I型细胞作为肺泡上皮的干细胞。体内损伤后II型细胞分化功能和增殖能力的研究因损伤全身反应的复杂性而受阻。体外研究又受到培养的分离II型细胞增殖潜能受损和分化标志物丧失的限制。我们描述了一种体外系统，其中II型细胞自发增殖并同时保持分化特征。其他研究者在注入琼脂糖后将成年肺切片培养长达9周。为了进一步开发该模型用于研究上皮细胞分化和增殖，我们评估了注入琼脂糖的大鼠肺切片外植体中的上皮分化、增殖能力和细胞特异性基因表达的调控。我们制备了1毫米厚的外植体，并将它们培养长达2周。通过光镜和电镜形态学、上皮细胞特异性表面活性物质蛋白的积累以及磷脂分析证实了分化的维持。通过测量基线时以及对生长刺激反应时肺泡和小气道细胞中[3H]胸腺嘧啶核苷掺入来评估增殖能力。糖皮质激素以剂量依赖性方式抑制II型细胞增殖。糖皮质激素对外植体中RNA水平的调节方式与体内和胎儿肺外植体中所见相似。成年肺切片外植体中的肺泡上皮维持分化功能和增殖能力，从而为研究远端气道和肺泡细胞内稳态及对损伤的反应提供了一个有用的系统。
