Análise da expressão de desmogleína 1 e desmoplaquina nas linhagens de células epiteliais MDCK e MCF-7 antes e após uma transição fenotípica de crescimento

• Autor(a) principal: França, Andressa Antunes Prado de
• Data de Publicação: 2008
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2357
• topic: Desmogleína; Cultura de células; Padrões de crescimento; Desmoglein; Cells culture; Growth pattern; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

Previous studies have identified an alteration in the growth pattern of some established cell lines cultured in monolayer, like MDCK cells, derived from normal canine kidney epithelium, and MCF-7 cells, obtained from human breast carcinoma. On MDCK cells, a transition from an exponential behavior to a power-law distribution was observed. On MCF-7 cells, the observed transition behaved the opposite way, changing from a power law to an exponential decay. In the present study, the expression of the adhesion protein desmoglein (Dsg) was analysed by western blot and immunofluorescence, using samples from the mentioned cell lines before and after the growth phenotypic transition. Thus, samples from MDCK cells were taken before 76 hours and after 146 hours from platting, and samples from MCF-7 cells, before 64 hours and after 140 hours, for the analysis of the desmosomal cadherin. After obtaining the four samples, they had their protein profile characterized by one dimension electrophoresis in poliacrilamide gel 8%, following which the proteins were transferred to a nitrocellulose membrane, by electrophoretic transfer, and the presence of desmogleína was revealed with a solution from the monoclonal antibody 32-2B, specific to isoform 1. The results from the western blot indicate that the samples from MCF-7 presented higher expression of Dsg1 than MDCK. MCF-7 before the transition had the highest expression of Dsg1, and between the samples of MDCK, the one obtained after the transition showed higher expression of Dsg1. In order to perform the immunofluorescence, samples from the two cell lines were obtained in the same time mentioned before. The cells had been platted onto 13mm glass coverslips in 24-well plates, being fixed and marked with primary 11-5F antibody to another desmosome protein, desmoplakin, and then with secondary antibody marked with FITC. The results from immunofluorescence indicate higher concentration of desmosomal proteins in the cell membrane of MCF-7 cells before the transition, in relation to the sample from after the phenotypic growth transition, and in MCDK cells, more intense marking was seen after the transition, considering that the MCF-7 cell presented higher marking than MDCK for desmosomal proteins, in accordance with the results from the western blot. The results from this experiment indicate a relation between the level of expression of Dsg and the growth pattern into the studied cell lines, in such a way that a possible role of this protein in cell cycle control pathways should be further discussed.