Assay for establishment of highly polarized retinal pigment epithelial cells

本课题组试图建立一种可重复性的高极化RPE的培养方法，并试图利用极化培养的RPE来阐明视网膜疾病的病理生理机制。T75培养瓶原代培养的RPE细胞接种于纤维连接蛋白包被的TrswellTM。RPE细胞在1%胎牛血清中培养4周以上。与天然组织一样，猪RPE细胞形成单层，着色良好，呈规则的六角形排列。TJ蛋白ZO-1的表达、微绒毛的闭塞和发育、色素在顶侧的定位、基底侧的核和紧密连接的复合体的存在证实了RPE层的完整性。分化良好的RPE细胞优先分泌血管内皮生长因子-A到组织的基底外侧。在极化培养RPE系统的应用方面，经肿瘤坏死因子-α刺激后，24小时时比对照组减少了约15%，这种作用可被抗肿瘤坏死因子-α抗体所消除。这种还原作用可被SB203580抑制剂所抑制，但不能被其他任何抑制剂所抑制。

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 TranswellTM. 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.