Kelly S P, Wood C M
Department of Biology, McMaster University, Hamilton, Ontario, Canada, L8S 1S1.
J Membr Biol. 2002 Nov 1;190(1):29-42. doi: 10.1007/s00232-002-1020-x.
Procedures for the preparation and culture of branchial epithelia from dispersed gill cells of freshwater tilapia (Oreochromis niloticus) are described. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on both the apical and basolateral side with isotonic media containing 6% fetal bovine serum (FBS). When the apical medium was replaced with freshwater (pseudo in vivo asymmetrical culture conditions), transepithelial resistance (TER) increased markedly, transepithelial potential became negative, and paracellular permeability decreased. The physiological effects of cortisol and 10% homologous (tilapia) serum were investigated. Tilapia serum (TS) was prepared from unstressed and stressed fish and therefore allowed comparison between the effects of homologous serum derived from fish in differing physiological states. Under both symmetrical and asymmetrical culture conditions, cortisol significantly elevated TER across cultured tilapia gill epithelia, indicative of a significant increase in epithelial "tightness." Cortisol reduced transepithelial Na + and Cl? movement and paracellular permeability. The glucocorticoid agonist dexamethasone elicited a similar response, which was inhibited by the glucocorticoid antagonist (receptor blocker) RU486. Cortisol did not stimulate active ion transport across epithelia under either symmetrical or asymmetrical culture conditions. In epithelia supplemented with TS from stressed fish, physiological changes in cultured preparations were consistent with those observed in FBS + cortisol-supplemented epithelia. Differences between the physiological status of epithelia supplemented with TS from unstressed and stressed fish could be abolished with RU486. Using TS as a medium supplement did not stimulate active ion transport under asymmetrical culture conditions, although Na +-K +-ATPase activity increased in TS-supplemented epithelia relative to FBS-supplemented preparations.
本文描述了从淡水罗非鱼（尼罗罗非鱼）分散的鳃细胞制备和培养鳃上皮的方法。上皮细胞在可渗透支持物（对苯二甲酸酯膜，“滤器”）上培养，并在顶端和基底外侧用含有6%胎牛血清（FBS）的等渗培养基进行灌流。当用淡水替换顶端培养基（模拟体内不对称培养条件）时，跨上皮电阻（TER）显著增加，跨上皮电位变为负值，细胞旁通透性降低。研究了皮质醇和10%同源（罗非鱼）血清的生理作用。罗非鱼血清（TS）取自未受应激和受应激的鱼，因此可以比较不同生理状态下鱼源同源血清的作用。在对称和不对称培养条件下，皮质醇均显著提高了培养的罗非鱼鳃上皮的TER，表明上皮“紧密性”显著增加。皮质醇减少了跨上皮Na⁺和Cl⁻的移动以及细胞旁通透性。糖皮质激素激动剂地塞米松引发了类似的反应，该反应被糖皮质激素拮抗剂（受体阻滞剂）RU486抑制。在对称或不对称培养条件下，皮质醇均未刺激跨上皮的主动离子转运。在用受应激鱼的TS补充的上皮细胞中，培养制剂的生理变化与在补充FBS+皮质醇的上皮细胞中观察到的变化一致。用RU486可以消除补充未受应激和受应激鱼的TS的上皮细胞生理状态之间的差异。在不对称培养条件下，使用TS作为培养基补充剂不会刺激主动离子转运，尽管相对于补充FBS的制剂，补充TS的上皮细胞中Na⁺-K⁺-ATP酶活性增加。
