Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVFE) lines for use in appropriate model systems. We steadily transfected a retroviral construct containing human telomerase reverse transcriptase (hTERT) into primary normal hVFE to establish a continuously replicating hVFE cell line. Immortalized hVFE across passages have cobblestone morphology, express epithelial markers cytokeratin 4, 13 and 14, induced hTERT gene and protein expression, have similar RNAseq profiling, and can continuously grow for more than 8 months. DNA fingerprinting and karyotype analysis demonstrated that immortalized hVFE were consistent with the presence of a single cell line. Validation of the hVFE, in a three-dimensional in vitro VF mucosal construct revealed a multilayered epithelial structure with VF epithelial cell markers. Wound scratch assay revealed higher migration capability of the immortalized hVFE on the surface of collagen-fibronectin and collagen gel containing human vocal fold fibroblasts (hVFF). Collectively, our report demonstrates the first immortalized hVFE from true VFs providing a novel and invaluable tool for the study of epithelial cell-fibroblast interactions that dictate disease and health of this specialized tissue.

中文翻译：

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新型永生化人声带上皮细胞系：粘膜生物学的体外工具

声带 (VF) 粘膜生物学的研究需要必要的人类声带上皮细胞 (hVFE) 系用于适当的模型系统。我们将含有人端粒酶逆转录酶 (hTERT) 的逆转录病毒结构稳定地转染到原代正常 hVFE 中，以建立连续复制的 hVFE 细胞系。跨代永生化 hVFE 具有鹅卵石形态，表达上皮标志物细胞角蛋白 4、13 和 14，诱导 hTERT 基因和蛋白表达，具有相似的 RNAseq 分析，可连续生长 8 个月以上。DNA 指纹图谱和核型分析表明，永生化的 hVFE 与单个细胞系的存在一致。在三维体外 VF 粘膜构建体中验证 hVFE 揭示了具有 VF 上皮细胞标记的多层上皮结构。伤口划痕试验揭示了永生化 hVFE 在含有人声带成纤维细胞 (hVFF) 的胶原蛋白-纤连蛋白和胶原凝胶表面上的更高迁移能力。总的来说，我们的报告展示了第一个来自真正 VF 的永生化 hVFE，为研究决定这种特殊组织的疾病和健康的上皮细胞 - 成纤维细胞相互作用提供了一种新颖且宝贵的工具。