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- Material Type
- 文書・図像類
- Author/Editor
- 園田, 祥三
- Author Heading
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- Alternative Title
- Assay for establishment of highly polarized retinal pigment epithelial cells
- Text Language Code
- jpn
- Subject Heading
- Target Audience
- 一般
- Note (General)
- 2010-2011年度科学研究費助成事業(科学研究費補助金(若手研究(B)))研究成果報告書 課題番号：22791674 研究代表者：園田祥三 (鹿児島大学大学院医歯学総合研究科助教)近年、Transwellを利用した網膜色素上皮細胞 (RPE)培養によって、細胞極性を示すRPE (polarized RPE) 培養が可能になっている。我々は、さらに培養法を改良し、ヒトおよびブタRPEで、細胞極性が高くより分化した培養法を確立した。極性RPEの特徴は、形態的には細胞表面に微絨毛がよく発達、tight junction, adherence junctionがよく発達し形態的に生体RPEときわめて類似。機能的には、trans epithelial resisatance (TER)値がブタで200Ω・cm2 以上)、permeability assayで自由な物質の透過が制御、さらにVEGFやPEDFの極性分泌が生体と同じであることを確認した。Our group attempted to establish a reproducible method for culturing highly polarized RPE and tried to elucidate the pathophysiological mechanism of retinal disease using polarized culture RPE. Highly polarized RPE cells were grown on Transwell^{TM} filters (12 mm, i.d). Primary cultures of RPE cells on T75 flask were seeded on fibronectin-coated Transwell^{TM}. RPE cells were cultured in 1% FBS for more than 4 weeks. As in native tissue, porcine RPE cells formed a monolayer, were well pigmented, and were arranged in a regular hexagonal array. The integrity of RPE layer was confirmed by expression of TJ proteins ZO-1 and occluding and well developed microvilli, localization of pigment on the apical side, nuclei on basal side, and presence of tight‐junctional complexes by electron microscopy. Well-differentiated RPE cells secreted VEGF-A preferentially to the basolateral side of the tissue. With respect to application of polarized culture RPE system, after stimulation with TNF-a, TER decreased about 15% of reduction in comparison to control at 24 hours, which was abolished by anti-TNF-a antibody. This reduction was inhibited by SB203580 inhibitor, but not by any other inhibitors.