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Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line.

Wang J, Hu G, Lin Z, He L, Xu L, Zhang Y - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.

ABSTRACT
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.

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Immunostaining and western blot analysis of ZYM-SIEC02 cell cultures.A. pSIECs and ZYM-SIEC02 cells were stained with cytokeratin 18 antibodies (green) and propidium iodide (red). B. pSIECs and ZYM-SIEC02 cells were stained with pan-cytokeratin antibodies (green) and propidium iodide (red). C. expression of cytokeratin 18, E-cadherin, SI, villin, ZO-1 and occludin were determined by western blot analysis. Cytokeratin 18, pan-cytokeratin, E-cadherin, SI, villin, ZO-1 and occludin were expressed in both pSIECs (at passage 3) and ZYM-SIEC02 cells (at passages 15, 55, and 90).
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pone-0110916-g002: Immunostaining and western blot analysis of ZYM-SIEC02 cell cultures.A. pSIECs and ZYM-SIEC02 cells were stained with cytokeratin 18 antibodies (green) and propidium iodide (red). B. pSIECs and ZYM-SIEC02 cells were stained with pan-cytokeratin antibodies (green) and propidium iodide (red). C. expression of cytokeratin 18, E-cadherin, SI, villin, ZO-1 and occludin were determined by western blot analysis. Cytokeratin 18, pan-cytokeratin, E-cadherin, SI, villin, ZO-1 and occludin were expressed in both pSIECs (at passage 3) and ZYM-SIEC02 cells (at passages 15, 55, and 90).

Mentions: ZYM-SIEC02 cells were characterized using immunofluorescence and western blot analysis. Immunostaining for cytokeratin indicated characteristic disposition of intermediate filaments, and intracellular expression of cytokeratin 18 (Fig. 2A) and pan-cytokeratin (Fig. 2B).


Characteristic and functional analysis of a newly established porcine small intestinal epithelial cell line.

Wang J, Hu G, Lin Z, He L, Xu L, Zhang Y - PLoS ONE (2014)

Immunostaining and western blot analysis of ZYM-SIEC02 cell cultures.A. pSIECs and ZYM-SIEC02 cells were stained with cytokeratin 18 antibodies (green) and propidium iodide (red). B. pSIECs and ZYM-SIEC02 cells were stained with pan-cytokeratin antibodies (green) and propidium iodide (red). C. expression of cytokeratin 18, E-cadherin, SI, villin, ZO-1 and occludin were determined by western blot analysis. Cytokeratin 18, pan-cytokeratin, E-cadherin, SI, villin, ZO-1 and occludin were expressed in both pSIECs (at passage 3) and ZYM-SIEC02 cells (at passages 15, 55, and 90).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206455&req=5

pone-0110916-g002: Immunostaining and western blot analysis of ZYM-SIEC02 cell cultures.A. pSIECs and ZYM-SIEC02 cells were stained with cytokeratin 18 antibodies (green) and propidium iodide (red). B. pSIECs and ZYM-SIEC02 cells were stained with pan-cytokeratin antibodies (green) and propidium iodide (red). C. expression of cytokeratin 18, E-cadherin, SI, villin, ZO-1 and occludin were determined by western blot analysis. Cytokeratin 18, pan-cytokeratin, E-cadherin, SI, villin, ZO-1 and occludin were expressed in both pSIECs (at passage 3) and ZYM-SIEC02 cells (at passages 15, 55, and 90).
Mentions: ZYM-SIEC02 cells were characterized using immunofluorescence and western blot analysis. Immunostaining for cytokeratin indicated characteristic disposition of intermediate filaments, and intracellular expression of cytokeratin 18 (Fig. 2A) and pan-cytokeratin (Fig. 2B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus