RESUMEN
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.
Asunto(s)
Ácido Ascórbico/farmacología , Quemaduras/patología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Expresión Génica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Adulto , Araquidonato 12-Lipooxigenasa/análisis , Araquidonato 12-Lipooxigenasa/efectos de los fármacos , Quemaduras/tratamiento farmacológico , Células Cultivadas , Estudios Transversales , Ciclooxigenasa 1/análisis , Ciclooxigenasa 1/efectos de los fármacos , Oxidasas Duales/análisis , Oxidasas Duales/efectos de los fármacos , Femenino , Glutatión Peroxidasa/análisis , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Transferasa/análisis , Glutatión Transferasa/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/análisis , Factores de Intercambio de Guanina Nucleótido/efectos de los fármacos , Humanos , Masculino , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/análisis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/efectos de los fármacos , Peroxirredoxinas/análisis , Peroxirredoxinas/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Valores de Referencia , Reproducibilidad de los Resultados , Piel/efectos de los fármacos , Piel/patología , Estadísticas no Paramétricas , Ubiquitina-Proteína Ligasas/análisis , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Adulto JovenRESUMEN
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.
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Adulto Joven , Ácido Ascórbico/farmacología , Quemaduras/patología , Expresión Génica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Valores de Referencia , Piel/patología , Araquidonato 12-Lipooxigenasa/análisis , Araquidonato 12-Lipooxigenasa/efectos de los fármacos , Quemaduras/tratamiento farmacológico , Células Cultivadas , Estudios Transversales , Estadísticas no Paramétricas , Ubiquitina-Proteína Ligasas/análisis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/análisis , Ciclooxigenasa 1/análisis , Ciclooxigenasa 1/efectos de los fármacos , Peroxirredoxinas/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Oxidasas Duales/análisis , Oxidasas Duales/efectos de los fármacos , Glutatión Peroxidasa/análisis , Glutatión Peroxidasa/efectos de los fármacosRESUMEN
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.
Asunto(s)
Diferenciación Celular/fisiología , Citometría de Flujo/métodos , Queratinocitos/citología , Células Madre/citología , Animales , Biomarcadores/análisis , Técnicas de Cultivo de Célula , Células Cultivadas , Protocolos Clínicos , Humanos , Piel/citologíaRESUMEN
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.
Asunto(s)
Humanos , Animales , Células Madre/citología , Queratinocitos/citología , Diferenciación Celular/fisiología , Citometría de Flujo/métodos , Piel/citología , Biomarcadores/análisis , Células Cultivadas , Protocolos Clínicos , Técnicas de Cultivo de CélulaRESUMEN
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.
Asunto(s)
Quemaduras/genética , Factor 7 de Crecimiento de Fibroblastos/farmacología , Expresión Génica/genética , Queratinocitos/efectos de los fármacos , Cicatrización de Heridas/genética , Adulto , Animales , Quemaduras/patología , Estudios de Casos y Controles , Células Cultivadas , Regulación hacia Abajo , Femenino , Humanos , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Piel/citologíaRESUMEN
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.
