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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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Detection of Telomerase activity.A. Expression of hTERT protein was detected by western blot analysis. Expression of hTERT was readily detected in ZYM-SIEC02 cells at passage 50 and 90, but was barely detectable in pSIECs at passage 3. The A549 cell line was used as a positive control. B. Silver staining method was used to detect telomerase activity. The laddering patterns indicated that ZYM-SIEC02 cells display higher telomerase activity as compared to pSIECs and A549 cell line.
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pone-0110916-g004: Detection of Telomerase activity.A. Expression of hTERT protein was detected by western blot analysis. Expression of hTERT was readily detected in ZYM-SIEC02 cells at passage 50 and 90, but was barely detectable in pSIECs at passage 3. The A549 cell line was used as a positive control. B. Silver staining method was used to detect telomerase activity. The laddering patterns indicated that ZYM-SIEC02 cells display higher telomerase activity as compared to pSIECs and A549 cell line.

Mentions: Telomerase activity in both pSIECs and ZYM-SIEC02 cells was detected by Western blot and a TRAP-silver staining Telomerase Detection Kit. There were no significant differences observed in the level of hTERT protein in ZYM-SIEC02 cells at early or late passage, but ZYM-SIEC02 cells exhibited higher hTERT expression and activity than pSIECs (Fig. 4A, B).


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)

Detection of Telomerase activity.A. Expression of hTERT protein was detected by western blot analysis. Expression of hTERT was readily detected in ZYM-SIEC02 cells at passage 50 and 90, but was barely detectable in pSIECs at passage 3. The A549 cell line was used as a positive control. B. Silver staining method was used to detect telomerase activity. The laddering patterns indicated that ZYM-SIEC02 cells display higher telomerase activity as compared to pSIECs and A549 cell line.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110916-g004: Detection of Telomerase activity.A. Expression of hTERT protein was detected by western blot analysis. Expression of hTERT was readily detected in ZYM-SIEC02 cells at passage 50 and 90, but was barely detectable in pSIECs at passage 3. The A549 cell line was used as a positive control. B. Silver staining method was used to detect telomerase activity. The laddering patterns indicated that ZYM-SIEC02 cells display higher telomerase activity as compared to pSIECs and A549 cell line.
Mentions: Telomerase activity in both pSIECs and ZYM-SIEC02 cells was detected by Western blot and a TRAP-silver staining Telomerase Detection Kit. There were no significant differences observed in the level of hTERT protein in ZYM-SIEC02 cells at early or late passage, but ZYM-SIEC02 cells exhibited higher hTERT expression and activity than pSIECs (Fig. 4A, B).

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