ИСТИНА

Abscisic acid (ABA) is a plant stress hormone synthesized not only by plant cells, but also various types of human cells. Exogenous ABA can stimulate the differentiation of normal and tumor cell lines, including skin cells. The search for natural regulators of keratinocyte differentiation is relevant for both medicine and cosmetology. Consequently, there is a need to develop and standardize laboratory test systems for rapid analysis of new, potentially beneficial substances. Unlike 2D cell culturing, 3D cell culturing more closely mimics in vivo physiological conditions. The aim of this work was to study the short-term and long-term effects of ABA on the parameters of differentiation in 2D and 3D model systems based on keratinocytes. We found that under 2D cultivation conditions, ABA (2 mM) increases both expression of ER stress genes (CHOP, GRP78) and the level of synthesis of loricrin in human immortalized keratinocytes HaCaT after just 24 h. After 2 and 6 days of ABA exposure (2 mM) we observed an increase in the number of keratin 10 positive cells. Spheroids consisting of keratinocyte and fibroblast mix develop a surface layer, consisting of 1-2 sheets of elongated tightly adjacent cells; the central part is represented by a loose mass of fibroblast-like cells. Following exposure to ABA (2 mM, 48 h), thickening of the surface layer is observed, associated with an increase in the number of cell sheets. Immunochemical staining of keratinocyte and fibroblast markers on whole mounts showed that keratinocyte markers (keratin 10, 14) are localized in the spheroid surface layer, and fibroblast markers (vimentin) – in the central part. However, these results were not replicated on cryosections of spheroids. Skin-equivalent model grown for 29-31 days, consisting of HaCaT keratinocytes and fibroblasts, develops a surface layer of 2-3 flattened cell sheets resembling the epidermis. However, no stratification, no gradient of differentiation markers and no signs of keratinization were detected. Beneath the epidermis there is a thick layer of extracellular matrix with vimentin-positive fibroblasts forming dermis equivalent; its thickness increases after exposure to ABA (2 mM, 14 days). Immunochemical staining of keratin 14 shows a significant increase in fluorescence intensity in ABA-treated cells. The shape of keratinocytes changes from flattened to rounded. These data may support dedifferentiation stimulated by long-term cultivation with exposure to ABA (19-20 days). In conclusion, the constructed skin equivalent has serious limitations, but can be used as a simplified test system to assess the effect of ABA and other agents on keratinocyte differentiation