Pathology Department, School of Dentistry, University of São Paulo, Brazil.
Dental Surgery Department, School of Dentistry, University of São Paulo, Brazil.
Arch Oral Biol. 2020 Sep;117:104780. doi: 10.1016/j.archoralbio.2020.104780. Epub 2020 Jun 2.
To investigate whether the process of primary gingival keratinocytes culture obtained from normal human gingiva modifies the expression of keratins (K) 10, K14, and K19.
Human gingival fragments were collected from healthy individuals in the same oral site. One part of the samples underwent an immunohistochemistry assay for K10, K14, and K19. The labeling in the epithelium was quantified using a semiautomated method. Another part was used for primary gingival keratinocytes isolation and growth in two-dimensional culture. These cells were also stained for K10, K14, and K19 using immunofluorescence and immunocytochemistry. Positive cells were counted, and the nuclei and cytoplasmatic labeling areas were quantified.
In the gingival tissue, a higher expression was found for K14 versus K10 (p < 0.001); K19 was negative in all samples. In gingival keratinocytes culture, K14 (89.6 %) had the highest expression with significant differences in relation to K10 (76.9 %, p < 0.01) and K19 (9.9 %, p < 0.01). The cells positive for K14 exhibited larger nuclei in comparison with K10 (p < 0.05) and K19 (p < 0.05), suggesting a more undifferentiated phenotype. K19 cells showed the largest cytoplasmatic labeling in relation to K10- (p < 0.05) and K14-positive (p < 0.05) cells.
The process of growth in culture of gingival keratinocytes maintained the expression pattern of K10 and K14 observed in gingival tissues. However, this method induces the expression of K19, suggesting a potential transformation of the keratin network presented in the gingival keratinocytes during the formation of a monolayer in vitro. This reflects the dynamics of cell differentiation.
研究从正常人牙龈中获得的原代牙龈角质形成细胞培养过程是否会改变角蛋白（K）10、K14 和 K19 的表达。
从同一口腔部位的健康个体中收集牙龈组织碎片。一部分样本进行 K10、K14 和 K19 的免疫组织化学检测。使用半自动方法对上皮中的标记进行定量。另一部分用于原代牙龈角质形成细胞的分离和二维培养。使用免疫荧光和免疫细胞化学对这些细胞进行 K10、K14 和 K19 的染色。对阳性细胞进行计数，并对细胞核和细胞质标记区域进行定量。
在牙龈组织中，K14 的表达高于 K10（p<0.001）；所有样本中 K19 均为阴性。在牙龈角质形成细胞培养中，K14（89.6%）的表达最高，与 K10（76.9%，p<0.01）和 K19（9.9%，p<0.01）相比有显著差异。与 K10（p<0.05）和 K19（p<0.05）相比，K14 阳性细胞的细胞核更大，提示更未分化的表型。与 K10-（p<0.05）和 K14 阳性（p<0.05）细胞相比，K19 细胞的细胞质标记最大。
在培养中生长的牙龈角质形成细胞保持了在牙龈组织中观察到的 K10 和 K14 的表达模式。然而，这种方法诱导了 K19 的表达，提示在体外形成单层过程中，牙龈角质形成细胞中存在的角蛋白网络发生了潜在的转化。这反映了细胞分化的动态。
