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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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Optimization of culture medium for ZYM-SIEC02 cells.A. ZYM-SIEC02 cells were cultured for 24 h with 2%, 5%, 10%, 15%, or 20% FBS. 5% FBS was found to be the optimal concentration. B. Addition of 5% FBS or ITS, and/or EGF to the cell medium individually, compared with DMEM/F12 alone; Addition of ITS or ITS/EGF enhanced cell growthsimilar to the addition of 5% FBS, while addition of EGF alone did not have a significant effect. C. ITS/EGF or ITS significantly enhanced cell growth with the use of cell culture medium containing 5% FBS. D. Addition of ITS, EGF, or EGF/ITS to DMEM/F12 had little effect on the proliferation rate compared to the addition to 5% FBS. * P□0.05 versus basal conditions.
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pone-0110916-g006: Optimization of culture medium for ZYM-SIEC02 cells.A. ZYM-SIEC02 cells were cultured for 24 h with 2%, 5%, 10%, 15%, or 20% FBS. 5% FBS was found to be the optimal concentration. B. Addition of 5% FBS or ITS, and/or EGF to the cell medium individually, compared with DMEM/F12 alone; Addition of ITS or ITS/EGF enhanced cell growthsimilar to the addition of 5% FBS, while addition of EGF alone did not have a significant effect. C. ITS/EGF or ITS significantly enhanced cell growth with the use of cell culture medium containing 5% FBS. D. Addition of ITS, EGF, or EGF/ITS to DMEM/F12 had little effect on the proliferation rate compared to the addition to 5% FBS. * P□0.05 versus basal conditions.

Mentions: To optimize the culture conditions of our newly immortalized epithelial cells, medium supplements for growth of the cell line were tested. Based on cell growth responses, a concentration of 5% fetal bovine serum (FBS) was found to be optimal (Fig. 6A). To further confirm whether medium additives promote cell proliferation, 5% FBS or ITS (1.0 g/l insulin, 0.55 g/l transferrin, and 0.67 mg/l selenium) and/or epidermal growth factor (EGF, 10 ng/ml) were added to the culture medium. We found that, ITS alone or a combination of ITS and EGF enhanced cell growth equivalent to that induced by 5% FBS alone (Fig. 6B). Moreover, the addition of ITS to the culture medium containing 5% FBS further promoted cell proliferation while EGF alone had no effect (Fig. 6C, D).


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)

Optimization of culture medium for ZYM-SIEC02 cells.A. ZYM-SIEC02 cells were cultured for 24 h with 2%, 5%, 10%, 15%, or 20% FBS. 5% FBS was found to be the optimal concentration. B. Addition of 5% FBS or ITS, and/or EGF to the cell medium individually, compared with DMEM/F12 alone; Addition of ITS or ITS/EGF enhanced cell growthsimilar to the addition of 5% FBS, while addition of EGF alone did not have a significant effect. C. ITS/EGF or ITS significantly enhanced cell growth with the use of cell culture medium containing 5% FBS. D. Addition of ITS, EGF, or EGF/ITS to DMEM/F12 had little effect on the proliferation rate compared to the addition to 5% FBS. * P□0.05 versus basal conditions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110916-g006: Optimization of culture medium for ZYM-SIEC02 cells.A. ZYM-SIEC02 cells were cultured for 24 h with 2%, 5%, 10%, 15%, or 20% FBS. 5% FBS was found to be the optimal concentration. B. Addition of 5% FBS or ITS, and/or EGF to the cell medium individually, compared with DMEM/F12 alone; Addition of ITS or ITS/EGF enhanced cell growthsimilar to the addition of 5% FBS, while addition of EGF alone did not have a significant effect. C. ITS/EGF or ITS significantly enhanced cell growth with the use of cell culture medium containing 5% FBS. D. Addition of ITS, EGF, or EGF/ITS to DMEM/F12 had little effect on the proliferation rate compared to the addition to 5% FBS. * P□0.05 versus basal conditions.
Mentions: To optimize the culture conditions of our newly immortalized epithelial cells, medium supplements for growth of the cell line were tested. Based on cell growth responses, a concentration of 5% fetal bovine serum (FBS) was found to be optimal (Fig. 6A). To further confirm whether medium additives promote cell proliferation, 5% FBS or ITS (1.0 g/l insulin, 0.55 g/l transferrin, and 0.67 mg/l selenium) and/or epidermal growth factor (EGF, 10 ng/ml) were added to the culture medium. We found that, ITS alone or a combination of ITS and EGF enhanced cell growth equivalent to that induced by 5% FBS alone (Fig. 6B). Moreover, the addition of ITS to the culture medium containing 5% FBS further promoted cell proliferation while EGF alone had no effect (Fig. 6C, D).

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