Mießler Katharina S, Vitzthum Constanze, Markov Alexander G, Amasheh Salah
Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg 19b, 14163, Berlin, Germany.
Department of General Physiology, St. Petersburg State University, Universitetskaya nab. 7/9, Saint Petersburg, Russia, 199034.
Cytotechnology. 2018 Apr;70(2):567-576. doi: 10.1007/s10616-017-0130-3. Epub 2017 Aug 29.
Mammary gland epithelium is physiologically exposed to variations of hydrostatic pressure due to accumulation of milk and removal by suckling and mechanical milking. Integrity of the mammary gland epithelium primarily relies on the tight junction. To analyze pressure-induced effects on the tight junction, we established a modified Ussing chamber and tested the hypothesis if hydrostatic pressure on the basal side of the epithelium is able to affect barrier properties in a mammary epithelial cell model, in vitro. Therefore, a conventional Ussing chamber was modified by an additional tube system to apply hydrostatic pressure. Monolayers of the mammary epithelial cell line HC11 were mounted in the modified Ussing chambers and incubated with increasing basal hydrostatic pressure. Transepithelial resistance and short circuit current were recorded and compared to controls. Hydrostatic pressure was stably applied and incubation steps of 30 min were technically feasible, leading to a decrease of transepithelial resistance and an increase of short circuit current in all monolayers. In a series of experiments simulating the physiological exposure time by short intervals of 5 min, these electrophysiological findings were also observed, and monolayer integrity was not significantly perturbed as analyzed by fluorescence immunohistochemistry selectively staining tight junction proteins. Moreover, electrophysiology demonstrated reversibility of effects. In conclusion, the modified Ussing chamber is an adequate method to analyze the effects of hydrostatic pressure on epithelial cell monolayers, in vitro. Both, the reduction of transepithelial resistance and the increase of short circuit current may be interpreted as protective reactions.
乳腺上皮在生理上会因乳汁的积聚、哺乳和机械挤奶导致的排出而受到静水压力变化的影响。乳腺上皮的完整性主要依赖于紧密连接。为了分析压力对紧密连接的影响，我们建立了一个改良的尤斯灌流小室，并在体外乳腺上皮细胞模型中测试上皮基底侧的静水压力是否能够影响屏障特性这一假设。因此，通过一个额外的管道系统对传统的尤斯灌流小室进行了改良，以施加静水压力。将乳腺上皮细胞系HC11的单层细胞置于改良的尤斯灌流小室中，并在增加的基底静水压力下孵育。记录跨上皮电阻和短路电流，并与对照组进行比较。静水压力能够稳定施加，30分钟的孵育步骤在技术上是可行的，这导致所有单层细胞的跨上皮电阻降低，短路电流增加。在一系列通过5分钟短间隔模拟生理暴露时间的实验中，也观察到了这些电生理结果，并且通过选择性染色紧密连接蛋白的荧光免疫组织化学分析表明单层细胞的完整性没有受到显著干扰。此外，电生理学证明了这些效应具有可逆性。总之，改良的尤斯灌流小室是一种在体外分析静水压力对上皮细胞单层影响的合适方法。跨上皮电阻的降低和短路电流的增加都可以被解释为保护性反应。
