Expressão de genes relacionados à via de sinalização dos receptores TollLike em queratinócitos cultivados de pacientes com queimadura extensa
Arquivos
Data
2015
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
INTRODUÇÃO: A queimadura extensa pode evoluir com infecção grave
e sepse com aumento da morbidade e mortalidade. Ocorre um estado
contínuo de inflamação e o nível sérico de citocinas é elevado. Os
receptores do tipo Toll-Like são importantes receptores de células do
sistema imune inato que reconhecem antígenos. Portanto, existe a
necessidade da obtenção de perfil da expressão gênica da pele em
queimaduras extensas para análise inicial. OBJETIVO: Avaliar o perfil de
expressão de genes relacionados às vias de receptores Toll-Like em
amostras de cultura primária de queratinócitos humanos epidérmicos de
pacientes com queimadura grave. MÉTODOS: Após a obtenção de
fragmentos de pele viáveis com e sem queimadura, a cultura de
queratinócitos foi iniciada pelo método enzimático utilizando dispase
(Sigma-Aldrich) e Tripsina (Sigma-Aldrich). Após o estabelecimento da
linhagem celular, estas células foram tratadas com QIAzol Reagent®
(Qiagen) para a extração de RNA total. Este foi quantificado e analisado
quanto à pureza para se obter cDNA para a análise da expressão de 84
genes específico de vias TLR de placas de PCR Arrays (SA Biosciences).
RESULTADOS: Após a análise da expressão dos genes verificou-se que
21% destes genes estavam expressos diferencialmente, dos quais 100%
foram reprimidos ou hiporregulados. Dentre estes, os seguintes genes
(vezes de diminuição): HSPA1A (-58), HRAS (-36), MAP2K3 (-23),
TOLLIP (-23), RELA (-18), FOS (-16), e TLR1 (-6,0). CONCLUSÃO: O
perfil da expressão gênica da pele de pacientes com grande queimadura
mostrou 21% de genes alterados, destes 100% apresentaram-se
hiporregulados.
INTRODUCTION: Large burn injury can progress to severe infection and sepsis with increased morbidity and mortality. There is a continuous state of inflammation and serum levels of cytokines are high. The Toll-like receptors are important receptors of the innate immune system that recognize antigens. Therefore, a need exists for obtaining the gene expression profile of the skin of extensive burns for initial analysis. PURPOSE: To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human primary epidermal keratinocytes (hKEp) of patients with severe burns. METHODS: After obtaining viable fragments of skin with and without burn injury, culture hKEp was initiated by the enzymatic method using Dispase (Sigma-Aldrich) and Trypsin (Sigma-Aldrich). These cells were treated with Trizol® (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). CONCLUSION: The gene expression profile of skin of patients with large burn injury showed 21% altered genes, and 100% of them were downregulated.
INTRODUCTION: Large burn injury can progress to severe infection and sepsis with increased morbidity and mortality. There is a continuous state of inflammation and serum levels of cytokines are high. The Toll-like receptors are important receptors of the innate immune system that recognize antigens. Therefore, a need exists for obtaining the gene expression profile of the skin of extensive burns for initial analysis. PURPOSE: To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human primary epidermal keratinocytes (hKEp) of patients with severe burns. METHODS: After obtaining viable fragments of skin with and without burn injury, culture hKEp was initiated by the enzymatic method using Dispase (Sigma-Aldrich) and Trypsin (Sigma-Aldrich). These cells were treated with Trizol® (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). CONCLUSION: The gene expression profile of skin of patients with large burn injury showed 21% altered genes, and 100% of them were downregulated.
Descrição
Citação
CORNICK, Sarita Mac. Expressão de genes relacionados à via de sinalização dos receptores TollLike em queratinócitos cultivados de pacientes com queimadura extensa. 2015. 107 f. Tese (Doutorado em Ciências) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2015.