Аннотация:Abscisic acid (ABA) is a plant stress hormone synthesized not only by plant cells, butalso various types of human cells. Exogenous ABA can stimulate the differentiation ofnormal and tumor cell lines, including skin cells. The search for natural regulators ofkeratinocyte differentiation is relevant for both medicine and cosmetology. Consequently,there is a need to develop and standardize laboratory test systems for rapid analysis ofnew, potentially beneficial substances. Unlike 2D cell culturing, 3D cell culturing moreclosely mimics in vivo physiological conditions. The aim of this work was to study theshort-term and long-term effects of ABA on the parameters of differentiation in 2D and3D model systems based on keratinocytes.We found that under 2D cultivation conditions, ABA (2 mM) increases both expressionof ER stress genes (CHOP, GRP78) and the level of synthesis of loricrin in humanimmortalized keratinocytes HaCaT after just 24 h. After 2 and 6 days of ABA exposure (2mM) 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 representedby a loose mass of fibroblast-like cells. Following exposure to ABA (2 mM, 48 h), thickeningof the surface layer is observed, associated with an increase in the number of cell sheets.Immunochemical staining of keratinocyte and fibroblast markers on whole mountsshowed that keratinocyte markers (keratin 10, 14) are localized in the spheroid surfacelayer, and fibroblast markers (vimentin) – in the central part. However, these results werenot replicated on cryosections of spheroids.Skin-equivalent model grown for 29-31 days, consisting of HaCaT keratinocytesand fibroblasts, develops a surface layer of 2-3 flattened cell sheets resembling theepidermis. However, no stratification, no gradient of differentiation markers and nosigns of keratinization were detected. Beneath the epidermis there is a thick layer ofextracellular matrix with vimentin-positive fibroblasts forming dermis equivalent; itsthickness increases after exposure to ABA (2 mM, 14 days). Immunochemical stainingof 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 supportdedifferentiation stimulated by long-term cultivation with exposure to ABA (19-20 days).In conclusion, the constructed skin equivalent has serious limitations, but can be usedas a simplified test system to assess the effect of ABA and other agents on keratinocytedifferentiation.
