Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells:: development of an alveolo-capillary barrier in vitro
Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells:: development of an alveolo-capillary barrier in vitro
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DOI：
10.1038/labinvest.3700081
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发表时间：
2004-06-01
影响因子：
5
通讯作者：
Kirkpatrick, CJ
中科院分区：
文献类型：
--
作者：
Hermanns, MI;Unger, RE;Kirkpatrick, CJ
我们建立了人远端肺上皮细胞和人微血管内皮细胞的共培养体系，以研究上皮和内皮在急性肺损伤的发病和恢复过程中在肺泡毛细血管屏障的细胞相互作用。其目的是确定功能性细胞连接的发展和形成类似于体内观察到的紧密上皮屏障的条件。体外共培养系统由单层人肺上皮细胞系（A549或NCI H441）和原代人肺微血管内皮细胞（HPMEC）组成，位于可渗透滤膜的相对两侧。A549在形成具有完整细胞-细胞连接的紧密上皮屏障方面未能显示出足够的分化。用地塞米松刺激，NCI H441和HPMEC的共培养物建立了接触抑制分化的单层，NCI H441显示紧密连接蛋白ZO-1和粘附连接蛋白E-钙粘蛋白的连续的、圆周的免疫染色。通过透射电子显微镜证实了具有典型连接结构的极化上皮细胞单层的产生。共培养10-12天后，地塞米松处理导致平均跨双层电阻（TER）值为500 Ω cm（2），并与亲水性渗透性标记物钠-荧光素的通量降低相关。此外，基底外侧分布的促炎细胞因子肿瘤坏死因子-α引起了显着降低TER值后，24小时的曝光。通过在24小时内去除细胞因子，可以将TER的这种降低重新建立到对照水平。因此，NCI H441与HPMEC的共培养系统应该是一个合适的体外模型系统，以检查急性肺损伤、感染性肺疾病和中毒性肺损伤的发病机制中的上皮和内皮相互作用。此外，它还可用于改善肺部药物输送技术，这也需要功能屏障。
We have established a coculture system of human distal lung epithelial cells and human microvascular endothelial cells in order to study the cellular interactions of epithelium and endothelium at the alveolocapillary barrier in both pathogenesis and recovery from acute lung injury. The aim was to determine conditions for the development of functional cellular junctions and the formation of a tight epithelial barrier similar to that observed in vivo. The in vitro coculture system consisted of monolayers of human lung epithelial cell lines (A549 or NCI H441) and primary human pulmonary microvascular endothelial cells (HPMEC) on opposite sides of a permeable filter membrane. A549 failed to show sufficient differentiation with respect to formation of a tight epithelial barrier with intact cell-cell junctions. Stimulated with dexamethasone, the cocultures of NCI H441 and HPMEC established contact-inhibited differentiated monolayers, with NCI H441 showing a continuous, circumferential immunostaining of the tight junctional protein, ZO-1 and the adherens junction protein, E-cadherin. The generation of a polarized epithelial cell monolayer with typical junctional structures was confirmed by transmission electron microscopy. Dexamethasone treatment resulted in average transbilayer electrical resistance (TER) values of 500 Omega cm(2) after 10-12 days of cocultivation and correlated with a reduced flux of the hydrophilic permeability marker, sodium-fluorescein. In addition, basolateral distribution of the proinflammatory cytokine tumour necrosis factor-alpha caused a significant reduction of TER-values after 24 h exposure. This decrease in TER could be re-established to control level by removal of the cytokine within 24 h. Thus, the coculture system of the NCI H441 with HPMEC should be a suitable in vitro model system to examine epithelial and endothelial interactions in the pathogenesis of acute lung injury, infectious lung diseases and toxic lung injury. In addition, it could be used to improve techniques of lung drug delivery that also requires a functional barrier.