Zech J C, Pouvreau I, Cotinet A, Goureau O, Le Varlet B, de Kozak Y
Laboratoire d'Immunopathologie de l'Oeil, Paris, France.
Invest Ophthalmol Vis Sci. 1998 Aug;39(9):1600-8.
These experiments were designed to study the effect of cytokines and nitric oxide (NO) on rat retinal pigment epithelial (RPE) cell tight junctions in vitro.
Cultures of confluent RPE cells from retinas of PVG rats (a strain susceptible to development of experimental uveitis) were prepared on filters and incubated with various stimulants. The function of the tight junction was evaluated by measuring the transepithelial electrical resistance (TER) of the cell monolayer and the passive permeation of [3H]inulin across confluent RPE cells. The morphology of the intercellular junctions was visualized by immunolocalization of the tight junction-associated protein zonula occludens-1 (ZO-1) and F-actin.
Seventy-two hours after plating, the RPE cell monolayer showed a mean TER level of 67.6+/-18.8 omega/cm2. A decrease in TER was observed after treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). The addition of tumor necrosis factor-alpha (TNF-alpha) accelerated the decrease of TER, whereas NG-monomethyl-L-arginine (L-NMMA) (an NO synthase [NOS] inhibitor) did not further modify the resistance decrease. In contrast, 3-morpholino-sydnonimine (SIN-1), a sydnonimine analog and NO donor, increased the TER. The variations of TER were correlated with the transepithelial fluxes of [3H]inulin and with tight junction morphologic changes of ZO-1 and F-actin immunostaining.
Incubation with LPS associated with IFN-gamma and TNF-alpha induces alterations of RPE tight junctions, whereas NO is involved in the maintenance of their integrity. Cytokines and NO production could play a role in regulation of the blood-ocular barrier function and of the development of ocular inflammation.
这些实验旨在研究细胞因子和一氧化氮（NO）对体外培养的大鼠视网膜色素上皮（RPE）细胞紧密连接的影响。
在滤膜上制备来自PVG大鼠（一种易发生实验性葡萄膜炎的品系）视网膜的汇合RPE细胞培养物，并与各种刺激物一起孵育。通过测量细胞单层的跨上皮电阻（TER）以及[3H]菊粉在汇合RPE细胞上的被动渗透来评估紧密连接的功能。通过紧密连接相关蛋白闭合蛋白-1（ZO-1）和F-肌动蛋白的免疫定位观察细胞间连接的形态。
接种72小时后，RPE细胞单层的平均TER水平为67.6±18.8Ω/cm2。用γ-干扰素（IFN-γ）和脂多糖（LPS）处理后观察到TER降低。添加肿瘤坏死因子-α（TNF-α）加速了TER的降低，而NG-单甲基-L-精氨酸（L-NMMA）（一种一氧化氮合酶[NOS]抑制剂）并未进一步改变电阻的降低。相反，3-吗啉代-西多胺（SIN-1），一种西多胺类似物和NO供体，增加了TER。TER的变化与[3H]菊粉的跨上皮通量以及ZO-1和F-肌动蛋白免疫染色的紧密连接形态变化相关。
与IFN-γ和TNF-α相关的LPS孵育诱导RPE紧密连接的改变，而NO参与维持其完整性。细胞因子和NO的产生可能在血眼屏障功能调节和眼部炎症发展中起作用。
