Department of Surgery, Division of Otolaryngology-Head & Neck Surgery, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.
Laryngoscope. 2019 Oct;129(10):E355-E364. doi: 10.1002/lary.27909. Epub 2019 Mar 8.
A current lack of methods for epithelial cell culture significantly hinders our understanding of the role of the epithelial and mucus barriers in vocal fold health and disease. Our first objective was to establish reproducible techniques for the isolation and culture of primary porcine vocal fold epithelial cells. Our second objective was to evaluate the functional significance of cell cultures using an in vitro exposure to an inflammatory cytokine.
Epithelial cells were isolated from porcine vocal folds and expanded in culture. Characterization of cultures was completed by immunostaining with markers for pan-cytokeratin (epithelial cells), vimentin (stromal cells), von Willebrand factor (endothelial cell), and MUC1 and MUC4 (mucin) glycoproteins. Established epithelial cell cultures were then exposed to the inflammatory cytokine tumor necrosis factor alpha (TNF-α) for 24-hours, and transcript expression of MUC1 and MUC4 was evaluated.
Reproducible, porcine vocal fold epithelial cell cultures, demonstrating cobblestone appearance characteristic of the typical morphology of epithelial cell cultures were created. Cells showed positive staining for pan-cytokeratin with limited expression of vimentin and von Willebrand factor. Epithelial cells also expressed MUC1 and MUC4. TNF-α significantly increased transcript expression of MUC4.
Here, we present the first report of successful culture of primary porcine vocal fold epithelial cells. Cultures will provide researchers with a valuable new in vitro tool to investigate vocal fold epithelium and mucus as well as the effects of common challenges, including inflammatory cytokines, on these barriers.
NA Laryngoscope, 129:E355-E364, 2019.
目前缺乏用于上皮细胞培养的方法，这极大地阻碍了我们对声带健康和疾病中上皮和黏液屏障作用的理解。我们的首要目标是建立可重复的原代猪声带上皮细胞分离和培养方法。我们的第二个目标是使用体外暴露于炎症细胞因子来评估细胞培养的功能意义。
从猪声带中分离上皮细胞并在培养中进行扩增。通过用细胞角蛋白（上皮细胞）、波形蛋白（基质细胞）、血管性血友病因子（内皮细胞）和 MUC1 和 MUC4（粘蛋白）糖蛋白的标志物进行免疫染色，对培养物进行特征描述。然后将成熟的上皮细胞培养物暴露于炎症细胞因子肿瘤坏死因子-α（TNF-α）24 小时，评估 MUC1 和 MUC4 的转录表达。
成功创建了可重复的猪声带上皮细胞培养物，这些培养物具有上皮细胞培养物典型形态的鹅卵石外观。细胞对细胞角蛋白呈阳性染色，波形蛋白和血管性血友病因子的表达有限。上皮细胞还表达 MUC1 和 MUC4。TNF-α显著增加了 MUC4 的转录表达。
在这里，我们首次成功报告了原代猪声带上皮细胞的培养。这些培养物将为研究人员提供一种有价值的新的体外工具，用于研究声带上皮和黏液，以及包括炎症细胞因子在内的常见挑战对这些屏障的影响。
无。喉科学，129：E355-E364，2019 年。
