Establishment of a cell model targeted to NFAT signal transduction pathway for preliminary screening of FK506-like immunosuppressants / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 759-765, 2005.
Artigo
em Chinês
| WPRIM
| ID: wpr-237077
ABSTRACT
To screen NFAT antagonistic drugs and research signal transduction pathway related to NFAT. Four recombinant vectors were constructed. Each consists of three tandem copies of the human IL-2 distal NFAT-AP1 binding site in the context of the minimal IL-2 enhancer, either the sequence from -326 - +46 or the sequence from -89 - +46 (containing only the TATA box), driving a luciferase reporter gene or a destabilized enhanced green fluorescence protein (d2EGFP) reporter gene, respectively. Transient transfection of Jurkat cells was achieved by electroporation with 5 - 10 microg of the above plasmid and one pulse at 200V, 65ms. Plasmid pEFBos-mNFAT1 constitutively expressing murine full length NFAT1 protein was used for transient cotransfection. The results showed that neither of non-stimulation nor PMA or ionomycin stimulation alone could activate the reporter gene except PMA plus ionomycin costimulation. Furthermore, overexpressed murine NFAT1 augmented the activation of either IL-2 promoter or NFAT-AP1 enhancer drived reporter gene compared to the endogenous did. However, the reporter gene expression was nearly completely inhibited by pretreatment for 1h with FK506 at 5 microg/mL and then stimulation for 6-12h with PMA plus ionomycin in the presence of FK506. These findings indicated that such a transient Jurkat cell model offered a potential platform for preliminary screening of FK506 or CsA-like immunosuppressive agents.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Farmacologia
/
Transdução de Sinais
/
Elementos Facilitadores Genéticos
/
Interleucina-2
/
Regiões Promotoras Genéticas
/
Tacrolimo
/
Células Jurkat
/
Proteínas de Fluorescência Verde
/
Avaliação Pré-Clínica de Medicamentos
/
Fatores de Transcrição NFATC
Tipo de estudo:
Estudo diagnóstico
/
Estudo prognóstico
/
Estudo de rastreamento
Limite:
Animais
/
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2005
Tipo de documento:
Artigo
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