RESUMO
Gene therapy has attracted attention as a potentially effective alternative to liver transplantation for the treatment of hepatic failure. We chose the C/EBPbeta gene, which plays vital roles in liver regeneration, as a candidate for gene therapy, and examined its effect on hepatocyte survival and the suppression of liver inflammation. C/EBPbeta gene overexpression significantly maintained hepatocyte viability during 12 days of the culture. Urea synthesis ability, which is a liver-specific function, in Adv-C/EBPbeta-infected hepatocytes was stably maintained during the culture, but the activity per cell was significantly lower than that in non-infected cells. On the contrary, DNA synthesis activity in Adv-C/EBPbeta-infected hepatocytes was significantly higher than that in non-infected cells. COX-2 was induced in Adv-C/EBPbeta-infected hepatocytes, and the addition of NS398, a specific inhibitor of COX-2, suppressed the viability-maintenance effect. COX-2 was thus shown to be involved in the survival effect of C/EBPbeta gene. The introduction of the C/EBPbeta gene into liver-damaged mice significantly suppressed the serum AST and ALT activities. These results indicate that C/EBPbeta appears to be a survival factor under stressful conditions, and the introduction of the gene has therapeutic function against liver injury.
Assuntos
Hepatócitos/citologia , Fígado/lesões , Adenoviridae/genética , Adenoviridae/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Bromodesoxiuridina/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Tetracloreto de Carbono/farmacologia , Núcleo Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Meios de Cultura/farmacologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , DNA/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Hepatócitos/metabolismo , Inflamação , Óperon Lac , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Nitrobenzenos/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Temperatura , Fatores de Tempo , Ureia/metabolismoRESUMO
A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.
Assuntos
Adenoviridae/genética , Apoptose , Hepatócitos/citologia , Tiorredoxinas/genética , Animais , Células Cultivadas , Citoproteção , Vetores Genéticos , Hepatócitos/metabolismo , Humanos , Fígado Artificial , Masculino , Necrose , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Esferoides Celulares , Tiorredoxinas/biossíntese , TransfecçãoRESUMO
To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.
