RESUMO
PURPOSE: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. METHODS: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. RESULTS: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). CONCLUSION: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.
Assuntos
Ácido Ascórbico/farmacologia , Queimaduras/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Adulto , Araquidonato 12-Lipoxigenase/análise , Araquidonato 12-Lipoxigenase/efeitos dos fármacos , Queimaduras/tratamento farmacológico , Células Cultivadas , Estudos Transversais , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 1/efeitos dos fármacos , Oxidases Duais/análise , Oxidases Duais/efeitos dos fármacos , Feminino , Glutationa Peroxidase/análise , Glutationa Peroxidase/efeitos dos fármacos , Glutationa Transferase/análise , Glutationa Transferase/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Humanos , Masculino , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/análise , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/efeitos dos fármacos , Peroxirredoxinas/análise , Peroxirredoxinas/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Reprodutibilidade dos Testes , Pele/efeitos dos fármacos , Pele/patologia , Estatísticas não Paramétricas , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Adulto JovemRESUMO
Abstract Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.
Assuntos
Humanos , Masculino , Feminino , Adulto , Adulto Jovem , Ácido Ascórbico/farmacologia , Queimaduras/patologia , Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Valores de Referência , Pele/patologia , Araquidonato 12-Lipoxigenase/análise , Araquidonato 12-Lipoxigenase/efeitos dos fármacos , Queimaduras/tratamento farmacológico , Células Cultivadas , Estudos Transversais , Estatísticas não Paramétricas , Ubiquitina-Proteína Ligases/análise , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/análise , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 1/efeitos dos fármacos , Peroxirredoxinas/análise , Reação em Cadeia da Polimerase em Tempo Real , Oxidases Duais/análise , Oxidases Duais/efeitos dos fármacos , Glutationa Peroxidase/análise , Glutationa Peroxidase/efeitos dos fármacosRESUMO
PURPOSE: To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. METHODS: Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. RESULTS: Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. CONCLUSION: Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.
Assuntos
Diferenciação Celular/fisiologia , Citometria de Fluxo/métodos , Queratinócitos/citologia , Células-Tronco/citologia , Animais , Biomarcadores/análise , Técnicas de Cultura de Células , Células Cultivadas , Protocolos Clínicos , Humanos , Pele/citologiaRESUMO
Abstract Purpose: To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. Conclusion: Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.
Assuntos
Humanos , Animais , Células-Tronco/citologia , Queratinócitos/citologia , Diferenciação Celular/fisiologia , Citometria de Fluxo/métodos , Pele/citologia , Biomarcadores/análise , Células Cultivadas , Protocolos Clínicos , Técnicas de Cultura de CélulasRESUMO
PURPOSE: To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. METHODS: Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. RESULTS: Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. CONCLUSION: There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.
Assuntos
Queimaduras/genética , Fator 7 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica/genética , Queratinócitos/efeitos dos fármacos , Cicatrização/genética , Adulto , Animais , Queimaduras/patologia , Estudos de Casos e Controles , Células Cultivadas , Regulação para Baixo , Feminino , Humanos , Masculino , Camundongos , Reação em Cadeia da Polimerase , Pele/citologiaRESUMO
ABSTRACT PURPOSE: To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. METHODS: Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. RESULTS: Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. CONCLUSION: There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.