Le-Bel Gaëtan, Cortez Ghio Sergio, Larouche Danielle, Germain Lucie, Guérin Sylvain L
Centre Universitaire d'Ophtalmologie-Recherche (CUO Recherche), CHU de Québec, Québec (Qc), Canada.
Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX, CHU de Québec-Université Laval Research Center, Québec (Qc), Canada.
Methods Mol Biol. 2019;1879:43-73. doi: 10.1007/7651_2018_153.
Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.
电泳迁移率变动分析和蛋白质免疫印迹分别是研究DNA-蛋白质相互作用和蛋白质表达的简单、高效且快速的方法。上皮细胞的原代培养和传代培养广泛用于组织工程替代物的生产，并且作为基因表达研究的模型正日益受到欢迎。在培养过程中保存干细胞对于生产高质量的替代物至关重要。然而，细胞传代次数的增加与它们增殖能力的下降相关，直至达到衰老。这个过程可能由核定位转录因子如Sp1和NFI表达的改变所介导，其表达已被证明是细胞黏附、迁移和分化所必需的。在我们最近的一些研究中，我们观察到组织学和结构特征较差的重建组织与其组成上皮细胞中Sp1低表达之间存在相关性。因此，监测这些转录因子在人皮肤和角膜上皮细胞中的表达和DNA结合情况是表征原代培养上皮细胞质量的有用工具。
