Fanucchi M V, Harkema J R, Plopper C G, Hotchkiss J A
Department of Pathology, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan 48824, USA.
Am J Respir Cell Mol Biol. 1999 Jun;20(6):1274-85. doi: 10.1165/ajrcmb.20.6.3451.
The surface epithelium lining the nasal airways is a potential target for inhaled contaminants such as ozone, endotoxin, formaldehyde, tobacco smoke, and organic dusts. The epithelial response to injury may depend on the toxicant, the type of epithelium, the severity of the injury, and the presence of inflammatory cells and their secreted products. To study mechanisms of toxicant-induced epithelial injury and repair, in the absence of cellular inflammation or other systemic effects, we have developed a culture system to maintain morphologically distinct nasal airway epithelium in vitro. Microdissected maxilloturbinates and proximal nasal septa of male F344/N rats were cultured at an air-liquid interface for up to 14 d in supplemented serum-free medium. Maxilloturbinates are lined by nonciliated cuboidal nasal transitional epithelium (NTE) with few or no mucous cells. The proximal nasal septum is lined by a mucociliary respiratory epithelium (RE) that normally contains numerous mucous cells. Preservation of the normal RE and NTE phenotype in culture was assessed by light and electron microscopy, and analysis of an airway mucin gene (rMuc-5AC) messenger RNA (mRNA). Both RE and NTE retained normal cell morphology for 14 d in culture (DIC). After 14 DIC there were 20% fewer RE cells in the septa (equal loss of ciliated and mucous cells) and 25% more NTE cells in the maxilloturbinates (increased number of basal cells). Compared with the RE, the NTE expressed consistently low levels of rMuc-5AC mRNA and had little to no histochemically detectable intraepithelial mucosubstances (IM) after 0, 3, 7, or 14 DIC. The amount of stored IM and the steady-state levels of rMuc-5AC mRNA in the RE decreased with time in culture. In summary, this culture system can maintain fully differentiated secretory and nonsecretory rat airway epithelia in vitro for up to 14 d. This study was an essential first step in developing a system to study the pathogenesis of toxicant-induced airway epithelial injury and mechanisms of cellular repair and adaptation in the absence of cellular inflammation and other systemic influences.
鼻腔气道内衬的表面上皮是吸入性污染物（如臭氧、内毒素、甲醛、烟草烟雾和有机粉尘）的潜在靶点。上皮对损伤的反应可能取决于毒物、上皮类型、损伤的严重程度以及炎症细胞及其分泌产物的存在。为了在无细胞炎症或其他全身效应的情况下研究毒物诱导的上皮损伤和修复机制，我们开发了一种培养系统，以在体外维持形态上不同的鼻腔气道上皮。将雄性F344/N大鼠的显微切割的上颌鼻甲和近端鼻中隔在气液界面上，在补充了无血清培养基中培养长达14天。上颌鼻甲内衬无纤毛的立方状鼻腔过渡上皮（NTE），几乎没有或没有黏液细胞。近端鼻中隔内衬黏液纤毛呼吸上皮（RE），通常含有大量黏液细胞。通过光镜和电镜以及气道黏蛋白基因（rMuc-5AC）信使核糖核酸（mRNA）分析来评估培养物中正常RE和NTE表型的保存情况。RE和NTE在培养14天（相差显微镜）时均保持正常细胞形态。培养14天后，鼻中隔中的RE细胞减少20%（纤毛细胞和黏液细胞等量减少），上颌鼻甲中的NTE细胞增加25%（基底细胞数量增加）。与RE相比，NTE在培养0、3、7或14天后，rMuc-5AC mRNA表达水平持续较低，几乎没有或没有组织化学可检测到的上皮内黏液物质（IM）。培养过程中，RE中储存的IM量和rMuc-5AC mRNA的稳态水平随时间下降。总之，该培养系统可在体外将完全分化的分泌性和非分泌性大鼠气道上皮维持长达14天。这项研究是开发一个系统以研究毒物诱导的气道上皮损伤的发病机制以及在无细胞炎症和其他全身影响情况下细胞修复和适应机制的重要第一步。
