RESUMEN
Human DIXDC1 is a member of Dishevelled-Axin (DIX) domain containing gene family which plays important roles in Wnt signaling and neural development. In this report, we first confirmed that expression of Ccd1, a mouse homologous gene of DIXDC1, was up-regulated in embryonic developing nervous system. Further studies showed that Ccd1 was expressed specifically in neurons and colocalized with early neuronal marker Tuj1. During the aggregation induced by RA and neuronal differentiation of embryonic carcinoma P19 cells, expressions of Ccd1 as well as Wnt-1 and N-cadherin were dramatically increased. Stable overexpression of DIXDC1 in P19 cells promoted the neuronal differentiation. P19 cells overexpressing DIXDC1 but not the control P19 cells could differentiate into Tuj1 positive cells with RA induction for only 2 days. Meanwhile, we also found that overexpression of DIXDC1 facilitated the expression of Wnt1 and bHLHs during aggregation and differentiation, respectively, while inhibited gliogenesis by down-regulating the expression of GFAP in P19 cells. Thus, our finding suggested that DIXDC1 might play an important role during neurogenesis, overexpression of DIXDC1 in embryonic carcinoma P19 cells promoted neuronal differentiation, and inhibited gliogenesis induced by retinoic acid.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Neurogénesis/efectos de los fármacos , Neuroglía/citología , Neuronas/citología , Neuronas/efectos de los fármacos , Tretinoina/farmacología , Animales , Línea Celular Tumoral , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reguladores , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Neuroglía/efectos de los fármacosRESUMEN
AIM: By using of Escherichia coli DH5alpha to express GST-Ccd1 fusion protein and which was purified by affinity chromatographic separation. The purified target protein was used to immunize rabbits to prepare polyclonal antibody. METHODS: The previously constructed recombinant prokaryotic expression vector pGEX-5X-1-Ccd1 was transformed into Escherichia coli DH5alpha and was induced to expression by IPTG. The recombinant target protein was expressed with soluble state in Escherichia coli expression system which was separated and purified by chromatographic column stuffed with glutathione Sepharose 4B. The prepared antigen was used to immunize rabbits to get anti-Ccd1 specific rabbit original polyclonal antibody. RESULTS: ELISA data demonstrated that the antibody titer of the serum was up to 1:40 000. Immunohistochemistry analysis indicated that the "home-made" antibody had a specific interaction with Ccd1 protein and which could be used for extended experimental research. CONCLUSION: The anti-Ccd1 polyclonal antibody we had prepared had a high quality of potency and specificity. The antibody was demonstrated by experiments that which could totally fulfill the requirement of immunoblotting and immunohistochemistry study of Ccd1. The antibody provided an useful experimental tool to profoundly research the tissue expression profile, intercellular location and biological function of Ccd1.
