Wang Jing, Hu Guangdong, Lin Zhi, He Lei, Xu Lei, Zhang Yanming
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
PLoS One. 2014 Oct 22;9(10):e110916. doi: 10.1371/journal.pone.0110916. eCollection 2014.
The mucosal surface of intestine is continuously exposed to both potential pathogens and beneficial commensal microorganisms. Recent findings suggest that intestinal epithelial cells, which once considered as a simple physical barrier, are a crucial cell lineage necessary for maintaining intestinal immune homeostasis. Therefore, establishing a stable and reliable intestinal epithelial cell line for future research on the mucosal immune system is necessary. In the present study, we established a porcine intestinal epithelial cell line (ZYM-SIEC02) by introducing the human telomerase reverse transcriptase (hTERT) gene into small intestinal epithelial cells derived from a neonatal, unsuckled piglet. Morphological analysis revealed a homogeneous cobblestone-like morphology of the epithelial cell sheets. Ultrastructural indicated the presence of microvilli, tight junctions, and a glandular configuration typical of the small intestine. Furthermore, ZYM-SIEC02 cells expressed epithelial cell-specific markers including cytokeratin 18, pan-cytokeratin, sucrase-isomaltase, E-cadherin and ZO-1. Immortalized ZYM-SIEC02 cells remained diploid and were not transformed. In addition, we also examined the host cell response to Salmonella and LPS and verified the enhanced expression of mRNAs encoding IL-8 and TNF-α by infection with Salmonella enterica serovars Typhimurium (S. Typhimurium). Results showed that IL-8 protein expression were upregulated following Salmonella invasion. TLR4, TLR6 and IL-6 mRNA expression were upregulated following stimulation with LPS, ZYM-SIEC02 cells were hyporeponsive to LPS with respect to IL-8 mRNA expression and secretion. TNFα mRNA levels were significantly decreased after LPS stimulation and TNF-α secretion were not detected challenged with S. Typhimurium neither nor LPS. Taken together, these findings demonstrate that ZYM-SIEC02 cells retained the morphological and functional characteristics typical of primary swine intestinal epithelial cells and thus provide a relevant in vitro model system for future studies on porcine small intestinal pathogen-host cell interactions.
肠道黏膜表面持续暴露于潜在病原体和有益共生微生物中。最近的研究结果表明，肠道上皮细胞曾被视为一种简单的物理屏障，如今却是维持肠道免疫稳态所必需的关键细胞谱系。因此，建立一个稳定可靠的肠道上皮细胞系对于未来黏膜免疫系统的研究是必要的。在本研究中，我们通过将人端粒酶逆转录酶（hTERT）基因导入新生未哺乳仔猪的小肠上皮细胞，建立了猪肠道上皮细胞系（ZYM-SIEC02）。形态学分析显示上皮细胞片呈现出均匀的鹅卵石样形态。超微结构表明存在微绒毛、紧密连接以及典型的小肠腺状结构。此外，ZYM-SIEC02细胞表达上皮细胞特异性标志物，包括细胞角蛋白18、泛细胞角蛋白、蔗糖酶-异麦芽糖酶、E-钙黏蛋白和紧密连接蛋白1。永生化的ZYM-SIEC02细胞保持二倍体状态且未发生转化。此外，我们还检测了宿主细胞对沙门氏菌和脂多糖的反应，并通过感染鼠伤寒沙门氏菌（S. Typhimurium）验证了编码白细胞介素-8（IL-8）和肿瘤坏死因子-α（TNF-α）的mRNA表达增强。结果表明，沙门氏菌入侵后IL-8蛋白表达上调。脂多糖刺激后，Toll样受体4（TLR4）、Toll样受体6（TLR6）和白细胞介素-6（IL-6）的mRNA表达上调，ZYM-SIEC02细胞在IL-8 mRNA表达和分泌方面对脂多糖反应低下。脂多糖刺激后TNFα mRNA水平显著降低，用鼠伤寒沙门氏菌和脂多糖刺激均未检测到TNF-α分泌。综上所述，这些发现表明ZYM-SIEC02细胞保留了原代猪肠道上皮细胞的典型形态和功能特征，从而为未来猪小肠病原体与宿主细胞相互作用的研究提供了一个相关的体外模型系统。
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