期刊
EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES
卷 21, 期 1, 页码 77-86出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/S0928-0987(03)00205-7
关键词
Caco-2 cell model; immunohistochemistry; Gly-Sar uptake; hPEPT1; characterization
The aim of the present study was to investigate the influence of culture time on hPEPT1-mediated transport in Caco-2 cell monolayers. Peptide transport activity in Caco-2 cells grown in standard media and in a rapid 4-day model was first compared. The rapid 4-day Caco-2 cell model, cultured using a cocktail of growth factors and agonists, displayed lower peptide uptake capacity than Caco-2 cells grown for 4 days in conventional media, and was judged to be unsuitable for peptide transport studies. Peptide transport activity as well as monolayer integrity and tissue morphology were evaluated in the standard >21 days model as a function of the culture time. Peptide transport activity was studied using [C-14]-glycylsarcosine ([C-14]-Gly-Sar). Monolayer integrity was evaluated by transepithelial electrical resistance (TEER) measurements and [H-3]-mannitol permeabilities. Tissue morphology and hPEPT1 expression were studied using confocal laser scanning microscopy (CLSM) and conventional staining/immunostaining. Caco-2 cells grown in conventional media became confluent after 3-4 days. Mannitol permeability decreased from day 5 to 21 and TEER increased steadily until similar today 21. Apical hPEPT1 uptake activity appeared to be maximal in cells cultured for >21 days, whereas basolateral uptake reached a maximum already after 12 days in culture. In some of the passages studied, a secondary increase in hPEPT1 transport activity was observed in cells grown for >25 days. A large carrier-mediated transepithelial peptide flux component was evident from day 14. (C) 2003 Elsevier B.V. All rights reserved.
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