Peng Shaomin, Rahner Christoph, Rizzolo Lawrence J
Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Invest Ophthalmol Vis Sci. 2003 Feb;44(2):808-17. doi: 10.1167/iovs.02-0473.
The functional characteristics of tight junctions in the outer blood-retinal barrier change during embryonic development and in the presence of disease. A culture model of developing retinal pigment epithelium (RPE) was used to examine the regulation of the tight junctions.
RPE from chick embryos was cultured on filters that separated the apical and basal medium compartments. Cultures were maintained in various combinations of serum-free medium, serum-free medium that was conditioned by neural retinas, or serum-free medium that was supplemented with bovine pituitary extract, serum, or various hormones. Function was monitored by the transepithelial electrical resistance (TER) or the permeation of small organic tracers. Structure was monitored by immunofluorescence and freeze-fracture electron microscopy.
Functional analysis indicated differences in permeability among RPE of different embryonic age and culture conditions. In serum-free medium, the tight junctions were leaky or failed to form. Barrier properties increased if pituitary extract was added to the basal medium chamber or retina-conditioned medium was added to the apical chamber. Retina-conditioned medium was more effective at organizing tight junctional strands into a continuous network, but bovine pituitary extract appeared to modulate the permeability of that network. In combination, they synergistically elevated the TER to physiological levels. Although the thyroid hormone T3 had no effect, serum in the apical medium chamber inhibited the ability of RPE cells to respond to retina-conditioned medium.
Diffusible factors secreted by the neural retina acted synergistically with basolateral stimulation to regulate the structure and function of RPE tight junctions. Serum on the apical side of the RPE monolayer inhibited the ability of retinal factors to upregulate the tight junction barrier.
视网膜外血视网膜屏障紧密连接的功能特性在胚胎发育过程中以及疾病状态下会发生变化。本研究采用发育中的视网膜色素上皮(RPE)培养模型来研究紧密连接的调节机制。
将鸡胚的RPE细胞培养在分隔顶侧和基底侧培养基隔室的滤膜上。培养物分别用无血清培养基、经神经视网膜条件化的无血清培养基、添加牛垂体提取物、血清或各种激素的无血清培养基进行不同组合培养。通过跨上皮电阻(TER)或小分子有机示踪剂的渗透来监测功能。通过免疫荧光和冷冻蚀刻电子显微镜来监测结构。
功能分析表明,不同胚胎年龄和培养条件下的RPE细胞通透性存在差异。在无血清培养基中,紧密连接是渗漏的或未能形成。如果在基底侧培养基隔室中添加垂体提取物或在顶侧隔室中添加视网膜条件化培养基,屏障特性会增强。视网膜条件化培养基在将紧密连接链组织成连续网络方面更有效,但牛垂体提取物似乎能调节该网络的通透性。两者结合可协同将TER提高到生理水平。虽然甲状腺激素T3没有作用,但顶侧培养基隔室中的血清会抑制RPE细胞对视网膜条件化培养基的反应能力。
神经视网膜分泌的可扩散因子与基底外侧刺激协同作用,调节RPE紧密连接的结构和功能。RPE单层顶侧的血清会抑制视网膜因子上调紧密连接屏障的能力。