Rochwerger L, Dho S, Parker L, Foskett J K, Buchwald M
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
J Cell Sci. 1994 Sep;107 ( Pt 9):2439-48. doi: 10.1242/jcs.107.9.2439.
We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogen-coated filters was permeabilized with nystatin, a forskolin-stimulated Cl- permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogen-regulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them.
我们之前已经证明雌激素在体内可调节大鼠子宫上皮细胞中的囊性纤维化跨膜传导调节因子（CFTR）表达。本研究的目的是建立一个合适的体外系统，以研究类固醇激素对CFTR的调节作用。体外显示出高水平CFTR表达的大鼠子宫上皮细胞原代培养物，用腺病毒/猿猴病毒40（Adeno/SV40）病毒进行感染。分离并鉴定了一个克隆UIT 1.16，它保留了原代上皮细胞的形态，但增殖超过了原代培养的寿命。通过蛋白质免疫印迹法（western blots）中与SV40大T抗原相对应的条带的存在，以及它们持续增殖的能力，证实了UIT 1.16细胞成功永生化。透射电子显微镜研究表明，这些细胞保持了极化上皮细胞的特征，具有完善的膜结构域和特化的细胞间连接。当在改良的尤斯灌流小室（Ussing chambers）中检测细胞时，还观察到高跨上皮电阻。当在玻璃纤维涂层滤膜上生长的细胞的基底外侧细胞膜用制霉菌素通透后，在顶膜中观察到了福斯可林刺激的氯离子通透性，类似于其他表达CFTR的上皮细胞中的情况。UIT 1.16细胞在Northern印迹法中显示出高水平的CFTR表达。CFTR的表达依赖于培养基中雌激素的存在，因为当细胞在不含类固醇激素的血清培养基中培养时，几乎检测不到CFTR mRNA水平。然而，向该培养基中添加雌激素可防止CFTR mRNA的消失，证实了雌激素对UIT 1.16细胞系中CFTR表达的调节作用。新建立的UIT 1.16细胞系为分析类固醇激素对CFTR表达的调节提供了一个有价值的模型。此外，该细胞系还可用于研究正常雌性周期中CFTR在子宫中的作用，以及用于研究其他子宫上皮功能及其调节因子。
