RESUMEN
OBJECTIVE: To identify the expression of antagonist beta-TrCP protein in Sonic hedgehog signal transduction pathway and Wnt signal transduction pathway in hair follicle tissues. METHODS: The heads of day 18 embryo, and one day and six-days-old postnatal mice were acquired and treated with 40 g/L paraformaldehyde fixation for 48 h and paraffin embedding. The expression of beta-TrCP proteins was examined using LsAB (labelled streptavidin-biotin) method. RESULTS: beta-TrCP proteins were expressed in the cytoplasm of the hair stems of hair follicle, hair cuticle, cuticle of root sheath, Huxley's layer of internal root sheath cells, external root sheath and mesenchymal tissues, but not in connective tissue sheath and Henle's layer of internal root sheath. CONCLUSION: TrCP express in the developmental hair follicle tissues, which implicates that beta-TrCP regulate the developmental hair follicle by mediating the signal transduction pathways.
Asunto(s)
Folículo Piloso/metabolismo , Transducción de Señal , Proteínas con Repetición de beta-Transducina/biosíntesis , Proteínas con Repetición de beta-Transducina/fisiología , Animales , Animales Recién Nacidos , Folículo Piloso/embriología , Folículo Piloso/crecimiento & desarrollo , Inmunohistoquímica , Ratones , Factores de TiempoRESUMEN
OBJECTIVE: To construct recombinant adenovirus carrying the mouse dentin caveolin-1 gene using the recombinant adenoviral vector system AdEasy. METHODS: The cDNA fragment of caveolin-1 was derived from pTRE2-caveolin-1 by restriction enzyme digestion and subcloned into shuttle plasmid pAdtrack-CMV. The resulting plasmid pAdtrack-CMV-caveolin-1, after linearized by digesting with restriction endonuclease Pme I, was transformed into E. coli 1 BJ5183 which had been transformed by adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd-caveolin-1 was screened by kanamycin LB plate and then identified by restriction enzyme digestion. The linearized adenovirus plasmid pAd-caveolin-1 was packaged in 293 cells, then the recombinant adenovirus Ad-caveolin-1 was harvested. The expression of green fluorescence protein was observed under fluorescent microscope. With further amplification and purification, the titer of recombinant adenovirus was determined. RESULTS: The recombinant adenovirus was identified by restriction enzyme digestion analysis and gene sequencing. Cytopathic effect and the expression of GFP were observed in the infected 293 cells. The sequence of caveolin-1 gene insert was the same as that published in the GenBank. The titer of the recombinant adenovirus was 2 x 10(9) pfu/mL. CONCLUSION: The mouse caveolin-1 recombinant adenovirus was constructed successfully by using AdEasy adenovirus system. Cell transfection and expression of exogenous gene can be detected directly by observing the expression of GFP. These results provide the basis for the further study on the role of caveolin-1 gene in other scopes.
Asunto(s)
Adenoviridae/genética , Caveolina 1/genética , Animales , Caveolina 1/metabolismo , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
OBJECTIVE: The Sonic hedgehog signalling peptide has been demonstrated to play important roles in the growth and patterning of the tooth development. This study aims on whether the antagonist beta-transduction repeat containing protein of Sonic hedgehog signal transduction expressed in tooth germs ameloblast and odontoblast or not. METHODS: The mouse embryo heads of different developmental stages of E10.5, E13.5, E14.5, E16.5, E18.5 and P0, P3, P6 after birth were acquired fixed with 4% paraformaldehyde for 48 hours, embeded with Paraffin and examined using LsAB (labelled streptavidin-biotin) method to observe the beta-TrCP expression pattern in tooth germs, ameloblast and odontoblast. RESULTS: It was demonstrated that beta-TrCP expressed in oral epithelium, tooth bud, mesenchymal cell cytoplasm of ameloblast and odontoblast of different stage of tooth development. CONCLUSION: beta-TrCP expressed from early stage to later stage of murine tooth development. And these findings provide the evidence of antagonist regulatory pathways for shh in teeth development.