Van Winkle L S, Buckpitt A R, Plopper C G
Department of Anatomy, School of Veterinary Medicine, University of California-Davis 95616-8732, USA.
Am J Respir Cell Mol Biol. 1996 Jun;14(6):586-98. doi: 10.1165/ajrcmb.14.6.8652187.
Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary cytotoxicants and the progenitor during repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. The role of Clara cells in normal lung function is poorly understood partly because their abundance, sensitivity to cytotoxicants, and expression of differentiation markers vary by airway level and species. This study defines a strategy for maintenance in vitro of differentiated Clara cells within their local microenvironment. Lungs from adult mice were infalted with 1% agarose and distal airways were isolated by microdissection. Explants were cultured for 7 days in serum-free medium. Preservation of Clara cell morphology after 7 days in culture (DIC) was demonstrated using light and electron microscopy. Ciliated cells were also present. Cytochrome P450 monooxygenase activity, as measured by naphthalene epoxidation, was decreased 50% between 0 and 7 DIC, but the apparent stereoselectivity of metabolism was unchanged at 7 days. Marker proteins for differentiated Clara cells (secretory protein, CYP2F2 and CYP2B4) were detectable immunochemically throughout time in culture. Glutathione S-transferase activity and levels of reduced glutathione were unchanged over 7 DIC. We conclude that differentiated Clara cells can be maintained in cultures of explants from defined airway regions. Bronchiolar epithelial cells in this system are viable, synthesize and secrete secretory protein, metabolize xenobiotics via the cytochrome P450 system, have a stable phase II enzyme system, and maintain glutathione pools.
无纤毛细支气管上皮（克拉拉）细胞既是代谢活化的肺部细胞毒素的主要靶细胞，又是细支气管损伤后修复过程中的祖细胞，对于远端气道上皮功能和再生至关重要。克拉拉细胞在正常肺功能中的作用尚不清楚，部分原因是其丰度、对细胞毒素的敏感性以及分化标志物的表达因气道水平和物种而异。本研究定义了一种在其局部微环境中体外维持分化的克拉拉细胞的策略。用1%琼脂糖灌注成年小鼠的肺，并通过显微切割分离远端气道。外植体在无血清培养基中培养7天。使用光学和电子显微镜证明培养7天后（相差显微镜观察）克拉拉细胞形态得以保留。同时也存在纤毛细胞。通过萘环氧化测定的细胞色素P450单加氧酶活性在培养0至7天之间降低了50%，但在第7天时代谢的表观立体选择性未改变。在整个培养过程中都能通过免疫化学检测到分化的克拉拉细胞的标志物蛋白（分泌蛋白、CYP2F2和CYP2B4）。在7天的培养过程中，谷胱甘肽S-转移酶活性和还原型谷胱甘肽水平未发生变化。我们得出结论，分化的克拉拉细胞可以在来自特定气道区域的外植体培养物中维持。该系统中的细支气管上皮细胞是有活力的，能合成和分泌分泌蛋白，通过细胞色素P450系统代谢外源性物质，具有稳定的二期酶系统，并维持谷胱甘肽池。
