Immortalization of Fetal Bovine Colon Epithelial Cells by Expression of Human Cyclin D1, Mutant Cyclin Dependent Kinase 4, and Telomerase Reverse Transcriptase: An In Vitro Model for Bacterial Infection.
Immortalization of Fetal Bovine Colon Epithelial Cells by Expression of Human Cyclin D1, Mutant Cyclin Dependent Kinase 4, and Telomerase Reverse Transcriptase: An In Vitro Model for Bacterial Infection.
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DOI：
10.1371/journal.pone.0143473
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Fukuda T
中科院分区：
文献类型：
--
作者：
Kuroda K;Kiyono T;Isogai E;Masuda M;Narita M;Okuno K;Koyanagi Y;Fukuda T
牛是人类社会重要的经济动物。它们是生产牛奶和肉类等畜产品的关键。畜产品的生产效率受到人畜共患病原体感染的负面影响。为了预防和控制传染病，了解牛组织和病原菌之间的相互作用是很重要的。在这项研究中，我们建立了一个永生化的牛结肠上皮细胞系的体外感染模型，转导细胞与慢病毒载体含有基因编码的细胞周期调节因子细胞周期蛋白D1，突变型细胞周期蛋白依赖性激酶4（CDK 4），和人端粒酶逆转录酶（TERT）。建立的细胞系显示连续的细胞增殖，上皮标志物的表达，和一个完整的核型，表明细胞保持其原始性质的结肠来源的上皮。此外，我们将建立的细胞系暴露于肠道沙门氏菌和肠出血性大肠杆菌两种菌株。有趣的是，S.鼠伤寒沙门氏菌对已建立的细胞系具有更高的亲和性，并能侵入细胞质。肠道。定量RT-PCR显示Toll样受体1（TLR 1）、TLR 2和TLR 3基因在已建立的细胞中表达，而TLR 4、5和6基因在已建立的细胞中不表达。我们建立的永生化结肠上皮细胞应该是一个有用的工具，用于研究评估细菌感染的分子机制。
Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4), and human telomerase reverse transcriptase (TERT). The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1), TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection.