Modelo de pele humana reconstruída como plataforma para estudos de fotoenvelhecimento

Abstract

The effects of exposure to ultraviolet radiation on human skin have aroused interest in the scientific class, in understanding the transformations involved. Models of human skin reconstructed in vitro were developed as alternative methods to the use of animals in experimentation and proved to be useful and effective tools for studies. These models express different receptors, have proliferative capacity, extracellular matrix synthesis and metabolic functions, becoming tools of choice to understand the biological mechanisms of the skin. The validated and commercially available models are reconstructed from the cultivation of primary cells, facing some limitations in terms of acquisition and reproduction, which can affect their applicability. This work aimed to standardize a model of human skin reconstructed from permanent cell lines and evaluate the effects of Ultraviolet Radiation on this model. To this end, the work consisted of standardizing the model developed with permanent culture human keratinocytes and fibroblasts on a transwell-type membrane support for subsequent exposure to ultraviolet B radiation. For standardization, the cultivation times 10, 15 and 18 days were tested. Cell viability assays were performed using the MTT method and histological analysis by staining with hematoxylin and eosin. The condition that best represented the morphological structure of human skin, with epidermis and dermis, was 10 days of culture. The radiation doses used were 50mJ / cm2, 150mJ / cm2, 500mJ / cm2 and 1500mJ / cm2. At the lowest doses there was a small reduction in cell viability (50 and 150 mJ / cm2), the opposite occurred at doses of 500 and 1500mJ / cm2, showing a viability reduction of more than 50%. Histological sections showed a change in the epidermal layer of the skin after exposure, in a dose-dependent manner, visualizing the decrease in epithelial thickness, separation and vacuolization of cells in the epithelial layer. The proteolytic activity of MMPs 2 and 9 was assessed using a zymographic method. The lowest radiation dose triggered confirmed genotoxic activity, through the comet assay in alkaline pH. In view of the above, with this work it was possible to standardize a model of human skin reconstructed in vitro using lineage of permanent cells as a platform for photoaging studies using UVB radiation. Future experiments are needed to assess the potential for radiation damage and to elucidate the mechanisms involved, as well as the behavior of cells exposed in the three-dimensional microenvironment.