Year: 2021
Type: dissertation
Abstract: Aging represents a decline in physical and mental capacities, and consequently social, which culminates in the deterioration of the organism. Precisely because of its importance, it is necessary to understand its functioning and nature, and how it can affect organs such as the specific case of the skin.It is particularly important to find strategies that prevent, or at least delay skin aging, and that aim to reduce the risk of cancer and other diseases associated with aging, maintaining the physiological barrier and protection functions of the skin. Therefore, it is important to identify drugs capable of preventing or delaying skin aging, making it necessary to use in vitro models of senescence in skin cells, namely keratinocytes. Thus, the identification of experimental conditions that allow the induction of senescence in a spontaneously immortalized human keratinocyte cell line, which are applicable to the screening of hydrophilic and lipophilic compounds to identify potential anti-aging activity, was our focus of interest.The objectives of this work focused on evaluating the influence of some experimental conditions, namely the concentration of the inducing agent, cell density and the percentage of fetal bovine serum (FBS) in the cell culture medium in the induction of senescence in the spontaneously immortalized human keratinocyte line, HaCaT.For this, oxidative stress, caused by treatment of cell cultures with an oxidizing agent, H2O2, was used to induce cell senescence. As senescence markers, cell proliferation, β-Galactosidase activity, p53 and Lamin B1 expression, and the area and circularity of nuclei were evaluated, as well as the possible presence of senescence-associated heterochromatin foci (SAHF). In the initial phase of this study, the effect of different concentrations of H2O2 on cell metabolic activity as a function of cell density was evaluated after 48 hours, 72 hours or after 7 days. The results of these assays led us to select two cell densities, 0.5 and 0.25 x 105 cells/mL, cultured in the presence of 0.5 or 1% FBS, to proceed with the evaluation of the effect on cell senescence induction.Another variable tested was the effect of multiple additions of H2O2 throughout the culture period, that is, the reinforcement of the effect of H2O2 by repeated addition of a small concentration of this oxidant in the days following the first addition. Additionally, given that the optimization of an experimental model of keratinocyte senescence aims to enable the identification of drugs capable of preventing or at least delaying this process, the influence of treatment of cells with H2O2 in the presence of 0.1% DMSO, the vehicle most frequently used to dissolve lipophilic drugs.The results obtained did not allow the identification of culture and treatment conditions for HaCaT cells in which cell senescence induction was observed, since most of the evaluated markers did not show significant changes in the conditions tested. The most consistently observed marker of senescence was the decrease in cell proliferation in cells initially treated with 150 µM or 250 µM H2O2. Essentially the same results were verified irrespective of cell density, FBS concentration in the culture medium and the concentration of H2O2 added as a booster to the initial one.There was a increase in p53 protein in cells treated with 100 µM and 20 µM H2O2 boosters, not having the effect of other concentrations, although a trend of increase was observed with lower concentrations, up to 100 µM. An increase in Lamin B1 levels was also observed in cells initially treated with 50 µM H2O2 and 20 µM boosters.In none of the conditions tested, any alteration in nuclear morphology was observed, including the area and circularity of the nuclei, and the presence of SAHF was also not observed. Furthermore, under the conditions tested, the morphology, number and intensity of staining of cells with labelling due to SA-β-Gal activity were identical to those of untreated cells, the same happening in cells treated with vehicle (0.1% DMSO), in the absence and presence of the various concentrations and boosts of H2O2 and independently of cell density and FBS concentration in the culture medium.In short, the induction of cell senescence in vitro or mimicking in vitro aging is of great importance, due to the small number of models established to date and hence the need to establish experimental conditions that allow inducing the different characteristics of aging. The studies carried out did not allow us to identify the best conditions to use in a cell senescence model, but they brought us a little closer.
Author: Barbosa, Eliana Rita de Almeida
Language: Portuguese
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7224053059