Kaiser Bastian, Böttner Martina, Wedel Thilo, Brunner Ronald M, Goldammer Tom, Lesko Szilvia, Gäbel Gotthold, Gleich Alexander, Pfannkuche Helga
Institute of Veterinary Physiology, Faculty of Veterinary Medicine, Leipzig University, Leipzig, Germany.
Cells Tissues Organs. 2017;203(5):267-286. doi: 10.1159/000453394. Epub 2017 Jan 5.
Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.
连续细胞系已成为不可或缺的工具，通过提高实验可重复性和样本可用性，并减少实验动物的使用，从而推动了对细胞机制的研究。为了促进对猪结肠上皮屏障功能的研究，我们旨在建立一种具有延长复制能力的上皮细胞系。从一只3周龄仔猪的近端结肠分离细胞，并使用含有猿猴病毒40大T抗原（SV40 TAg）的重组逆转录病毒载体构建体进行转导。我们建立了一个克隆上皮细胞系，称为PoCo83-3，它稳定表达SV40 TAg，并在mRNA和蛋白质水平得到验证。PoCo83-3表现出上皮细胞特异性特征，如鹅卵石样形态、穹顶结构形成、顶端微绒毛的存在，以及角蛋白18、E-钙黏蛋白和紧密连接相关蛋白闭合蛋白-1、闭合蛋白和紧密连接蛋白-1的表达。为了验证PoCo83-3作为上皮屏障研究的体外模型，通过将细胞与TNF-α和IFN-γ孵育48小时，证明了促炎细胞因子诱导的屏障完整性改变。这些细胞因子处理导致跨上皮电阻降低。总之，PoCo83-3表现出延长的寿命和分化的表型，同时保持上皮特征。基于这些结果，我们将该细胞系作为研究猪结肠上皮屏障功能的有价值的体外模型。
