| 研究実績の概要 |
Although Caco-2 cell-based permeation assays are used to predict the oral absorption of drugs and phytochemicals in humans, they lack an important feature of the human intestine: the mucus layer on the luminal surface. We have developed a mucus-layered cell model using Caco-2 combined with the mucus-secreting HT29-MTX cell-line. Simultaneous seeding of Caco-2 and HT29-MTX cells at 8:2 and 9:1 ratios formed monolayers with good electric resistance and covered by a mucus layer, which we observed by scanning electron microscopy and fluorescence microscopy using Acridine orange. We prepared β-carotene micelles of different sizes containing PC, lysoPC as well as phospholipid-free micelles and performed permeation assays with Caco-2 and Caco-2/HT29-MTX monolayers cultured on Transwells. The new mucus-layered model could effectively detect the enhancement of β-carotene absorption by lysoPC (in a similar manner to the Caco-2 cell model) but contrary to the expected, permeability rates were generally higher in the Caco-2/HT29-MTX model, probably due to the weaker tight junctions in the HT29-MTX cells. To overcome this problem we cultured Caco-2 and Caco-2/HT29-MTX in conventional plates and performed carotenoid uptake assays. Uptake in the mucus-layered Caco-2/HT29-MTX model was clearly lower than in Caco-2 model when β-carotene was carried by larger micelles (lysoPC and phospholipid-free), but not when carried by very small PC micelles, suggesting that the mucus layer inhibited β-carotene uptake by intestinal cells, probably by slowing the permeation of larger mixed micelles.
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