RESUMO
Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.
Assuntos
Trombopoetina/metabolismo , Animais , Western Blotting , Células CHO , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Hematócrito , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Contagem de Plaquetas , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/fisiologia , Trombopoetina/genética , Trombopoetina/farmacologia , TransfecçãoRESUMO
Fibroblast cultures from wasted mice have been derived and the responses of these cultures to bleomycin treatment or gamma-irradiation have been examined. No differences were observed between wasted fibroblasts and littermate controls in the post-treatment inhibition of DNA replication. In contrast, a human SV-40 transformed ataxia telangiectasia fibroblast line mimicked the abnormal response of primary ataxia telangiectasia fibroblasts to gamma-rays or bleomycin and thus appears to be a useful in vitro model of ataxia telangiectasia.
