Construction of many tissues and organs de novo requires the use of external epithelial cell sources. In the present study, we optimized the isolation, expansion, and characterization of porcine oral epithelial cells from buccal tissue (Buccal Epithelial Cells, BECs). Additionally, we tested whether key markers [cytokeratin 14 (ck14), p63 protein, and sonic hedgehog molecule (shh)] expression profiles are correlated with three buccal epithelial clone types. Two digestion methods of BECs isolation [Method 1, M1 (collagenase IV/dispase and accutase) and Method 2, M2 (collagenase IV/dispase and trypsin/EDTA)] were compared. Cells obtained by more effective method were further cultured to the third passage and analyzed. Holoclone-, meroclone-, and paraclone-like colonies were identified based on BEC morphology. Immunofluorescent staining was performed to compare selected markers for the indicated buccal clone types. Comparative analysis demonstrated the advantage of isolation using M1 over M2. Cells from the third passage exhibited average 92.73% ± 2.27% presence of ck14. Real-time polymerase chain reaction confirmed expression of tested genes [cytokeratin 8 (ck8), ck14, integrin β1, and p63]. The highest level of ck14, shh and p63, was observed for holoclones. The comparable ck14 expression was observed in the mero- and paraclones. Meroclones expressed significantly lower levels of shh compared with paraclones. The weakest p63 expression was observed in the paraclone-like cells. It was demonstrated that holoclones are the richest in shh (+) and p63 (+) stem cells and these cells should appear to be a promising alternative for obtaining epithelial cells for tissue engineering purposes.

中文翻译：

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p63 蛋白和 Sonic Hedgehog 信号分子在口腔上皮全克隆中的表达增加

许多组织和器官的从头构建需要使用外部上皮细胞来源。在本研究中，我们优化了来自口腔组织（口腔上皮细胞，BEC）的猪口腔上皮细胞的分离、扩增和表征。此外，我们测试了关键标志物 [细胞角蛋白 14 (ck14)、p63 蛋白和声波刺猬分子 (shh)] 表达谱是否与三种口腔上皮克隆类型相关。比较了 BECs 分离的两种消化方法 [方法 1，M1（胶原酶 IV/分散酶和Accutase）和方法 2，M2（胶原酶 IV/分散酶和胰蛋白酶/EDTA）]。通过更有效的方法获得的细胞进一步培养至第三代并进行分析。基于 BEC 形态鉴定全克隆、meroclon 和类克隆样菌落。进行免疫荧光染色以比较指定的颊侧克隆类型的选定标记。比较分析证明了使用 M1 进行隔离优于 M2。第三代的细胞表现出平均 92.73% ± 2.27% 的 ck14 存在。实时聚合酶链反应证实了测试基因的表达 [细胞角蛋白 8 ( ck8 )、ck14、整合素 β1和p63 ]。在全克隆中观察到最高水平的 ck14、shh 和 p63。在 mero- 和 paraclones 中观察到类似的 ck14 表达。与副克隆相比，Meroclones 表达的 shh 水平显着降低。在类克隆细胞中观察到最弱的 p63 表达。已经证明，全克隆是最丰富的 shh (+) 和 p63 (+) 干细胞，这些细胞应该是获得用于组织工程目的的上皮细胞的有希望的替代品。