RESUMO
PURPOSE: To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences). RESULTS: After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.
Assuntos
Queimaduras/genética , Expressão Gênica , Queratinócitos/metabolismo , Receptores Toll-Like/genética , Adulto , Células Cultivadas , Epiderme/lesões , Epiderme/metabolismo , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , Receptores Toll-Like/metabolismo , CicatrizaçãoRESUMO
INTRODUCTION: At all stages of wound healing, growth factors and cytokines play a particularly important role in the interaction with keratinocytes cellular receptors. Keratinocytes have received little attention about their potential to act as a source and target of cytokines. Changes in the cytokine levels after the burning occur prior to the metabolic abnormalities. Thus, it may be possible to develop therapeutic interventions that can mitigate the acute inflammatory response and modulating expression of these cytokines. The objective was to evaluate the expression of 84 genes mediators of the inflammatory response by using PCR array in a primary human epidermal cultured keratinocytes from patients with burns. METHODS: Keratinocytes cultured from normal skin around injury from small and large burn patient were treated for DNA synthesis. The samples were analyzed by the PCR Superarray(®) assay and curve analyses were performed for 84 relevant human genes and their involvement in the inflammatory cytokines pathway and receptors. These genes were checked for the up or down regulation. And it was used MetaCore™ for the analysis of networks and Gene Ontology (GO) processes. RESULTS: Chemokines of the CXC family were more expressed in the large burn group, except CXCL12. The C, CC and CX3C chemokine family were downregulated, especially in the small burn group. The interleukins IL8 and IL1B were more expressed in large burn than in small burn; except IL13RA1, IL13 and IL5RA that were downregulated, mainly in the small burn group. CONCLUSIONS: The cytokine profile showed some important differences between the large and small burn patients, and from this original database, we can create new interventional trials in acute inflammation in burns.
Assuntos
Queimaduras/genética , Citocinas/genética , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Transcriptoma , Cicatrização/genética , Adulto , Queimaduras/imunologia , Estudos de Casos e Controles , Células Cultivadas , Quimiocinas C/genética , Quimiocinas C/imunologia , Quimiocinas CC/genética , Quimiocinas CC/imunologia , Quimiocinas CXC/genética , Quimiocinas CXC/imunologia , Citocinas/imunologia , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/imunologia , Mediadores da Inflamação/imunologia , Interleucina-13/genética , Interleucina-13/imunologia , Subunidade alfa1 de Receptor de Interleucina-13/genética , Subunidade alfa1 de Receptor de Interleucina-13/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Subunidade alfa de Receptor de Interleucina-5/genética , Subunidade alfa de Receptor de Interleucina-5/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Queratinócitos/imunologia , Masculino , Índice de Gravidade de Doença , Regulação para Cima , Cicatrização/imunologiaRESUMO
PURPOSE: To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences). RESULTS: After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome. .