Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Burn Injury Modulates the Expression Pattern of Oxidative Stress-Related Genes but not of Wound Healing-Related Genes in Human Keratinocytes
ALFREDO GRAGNANI, MD, PhD, Associate Professor, Silvana Aparecida Alves Correa de Noronha, PhD, Rafael de Moraes Petecof, BSc, Jessica Bonucci, Nursing, Samuel Marcos Ribeiro de Noronha, PhD, Lydia Masako Ferreira, MD, PhD, Chairwoman, Full Professor.
UNIVERSIDADE FEDERAL DE SÃO PAULO, São Paulo, Brazil.

Purpose: Burns caused by various types of agents has reached about one percent of world population. There are more than one million burns in the United States every year and about 5,000 of these injuries are fatal, making of burns the fourth leading cause of death from unintentional injuries in that country. The objective was to assess the expression profile of genes related to human Oxidative Stress (hOS) and human Wound Healing (hWH) in human epidermal keratinocytes from patients with severe burns.
Methods: Isolation of human epidermal keratinocytes were performed with fragments of viable skin from the margins of necrosis area of the burn removed by the surgeon (n=3). The separation of epidermis from dermis was initiated by the enzymatic method using dispase. Tissue was homogenized in Trizol reagent (Invitrogen). The RNA was dried and dissolved in RNase-free water. Total RNA was purified with Qiagen RNeasy Mini kit and subjected to DNAse treatment. The quantity and quality of extracted RNA were assessed by NanoVue Plus Spectrophotometer (GE Healthcare). Samples from each group were obtained to perform PCR Array plate containing 84 primers to study hOS and hWH pathways. The experiments were made in triplicates. Controls were healthy patients undergoing plastic surgery (n=3).
Results: After the expression analysis of the 84 studied genes for each biological function, we found, for hOS pathway, that 66% of these genes were differentially expressed, among these 89% were down regulated and 11% were up regulated. For this pathway some genes differentially expressed (p values were smaller than 0.05) attracted our attention specially for their antioxidant characteristics and also for genes involved in Reactive Oxygen Species (ROS) metabolism: ALB (-8.75), APOE (-8.75), ALOX12 (-5.05), DUOX1 (-6.48), DUOX2 (-4.18), MT3 (-8,75),GSR (-8.75), MTE (-8.75), SRXN1 (-7.91), TXNRD2 (-8.75), DUOX1 (-6.75), LYBB (-8.7), and PTGS2 (-8.16). Moreover, for hWH pathway we did not find significant differential expression for any of the 84 genes studied (p values were higher than 0.05).
Conclusions: Keratinocytes respond to burn injuries by decreasing the expression of genes related to antioxidants or involved in the Reactive Oxygen Species (ROS) metabolism. Our findings show that among the molecular changes caused by burn injuries, ROS related intracellular pathways are much more affected than wound healing related pathways in these cells. Furthermore, these data are in agreement with previous works investigating oxidative injury related genes (Castro et al., 2014; Oner et al., 2006; Toklu et al., 2006; Kabasakal et al., 2005). Oxidative stress determines changes in wound healing, and these two processes are fundamental to study new drugs to improve protocol of care of severe burned patient.
Acknowledgements: Dra. Andrea Fernandes Oliveira, coordinator of Burns Center of UNIFESP/EPM.
Support: FAPESP 2013/10905-0


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