RESUMO
OBJECTIVE: This study has aimed to study different culture systems that might stimulate an increase in cell proliferation of normal and osteoarthritis chondrocytes from articular cartilage in rat model. MATERIAL AND METHODS: Three culture systems using chondrocytes embedded in alginate beads were tested: chondrocytes cultured in Dulbecco's modified Eagle's medium (DMEM) as control, a co-culture system consisting of a monolayer of de-differentiated chondrocytes as a source of mitotic factors, and an enriched medium containing culture medium obtained from a monolayer of chondrocytes and DMEM. Normal and osteoarthritis chondrocytes were stained with 5-carboxyfluorescein diacetate succinimidyl ester and were cultured in each of the three systems. After 5 days of culture cell, proliferation was detected by flow cytometry. Chondrocyte phenotype was confirmed by collagen type II and MMP-3 expression. To determine possible molecules released into the medium by the cultured chondrocyte monolayer and which would probably be involved in cell proliferation, a study of mRNA and expression of transforming growth factor-beta1 (TGF-beta1), fibroblastic growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet derived growth factor-A (PDGF-A) and insulin-like growth factor-1 (IGF-1) proteins was conducted. RESULTS AND CONCLUSIONS: Chondrocytes in the co-culture system or in enriched medium showed an increase in proliferation; only when osteoarthritis chondrocytes were cultured in enriched medium would they display a statistically significant increase in their proliferation rate and in their viability. When chondrocytes from the monolayer were analysed, differential mRNA expression of TGF-beta1 and IGF-1 was found during all passages, which suggests that these two growth factors might be involved in chondrocyte proliferation.
Assuntos
Técnicas de Cultura de Células/métodos , Condrócitos/citologia , Alginatos , Animais , Cartilagem Articular/citologia , Cartilagem Articular/patologia , Desdiferenciação Celular , Proliferação de Células/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Técnicas de Cocultura/métodos , Colágeno Tipo II/metabolismo , Meios de Cultivo Condicionados/farmacologia , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica/genética , Ácido Glucurônico , Ácidos Hexurônicos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Mitose , Osteoartrite/patologia , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The type II voltage-dependent sodium channel is present in neuronal cells, where it mediates the propagation of nerve impulses. Restricted expression of the type II sodium channel gene to neurons is due, at least in part, to binding of the repressor protein REST (also termed NRSF or XBR) to the RE1 (also called NRSE) sequence in the type II sodium channel gene. Previous studies have shown that a domain in REST containing eight GL1-Krüppel zinc finger motifs mediates DNA binding. Deletional and GAL4-fusion gene analyses now reveal repressor domains that lie outside of the DNA-binding domain in both the amino and carboxyl termini of REST. Mutational analysis further identifies a single zinc finger motif in the carboxyl-terminal domain as being essential for repressing type II sodium channel reporter genes. These studies reveal two domains in REST that may mediate interactions with other proteins involved in restricting expression of a large set of genes to the vertebrate nervous system.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas Repressoras/genética , Canais de Sódio/genética , Fatores de Transcrição , Dedos de Zinco/genética , Análise Mutacional de DNA , Proteínas do Tecido Nervoso/biossíntese , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão , Deleção de Sequência , Canais de Sódio/biossíntese , Relação Estrutura-AtividadeRESUMO
Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells. We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element. Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST protein, whereas expression of a dominant negative form of REST in nonneuronal cells relieves silencing mediated by the native protein. REST transcripts in developing mouse embryos are detected ubiquitously outside of the nervous system. We propose that expression of the type II sodium channel gene in neurons reflects a default pathway that is blocked in nonneuronal cells by the presence of REST.
Assuntos
Regulação da Expressão Gênica , Sistema Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Repressoras/biossíntese , Canais de Sódio/biossíntese , Fatores de Transcrição/biossíntese , Sequência de Aminoácidos , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Desenvolvimento Embrionário e Fetal , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Sistema Nervoso/embriologia , Especificidade de Órgãos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de ZincoRESUMO
Sequences front a cDNA of dengue virus type 4 were cloned into transcription vectors. These sequences included the E, NS1, NS2A, NS2B, NS3 genes. RNA transcripts produced in vitro from these plasmids were used in hybridization assays to detect dengue viral sequences. With these RNA-probes we have been able to detect molecules of serotype-specific dengue 4 viral RNA. Moreover, the riboprobes detected viral sequences of other serotypes in the following order of sensitivity 4 > 2 > 3 > 1, and might be useful to differentiate serotypes.
