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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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Morphological features of pSIECs and ZYM-SIEC02 cells.A. Cellular morphology of pSIECs at 7 days in culture (100×) and at passage 5 (100×); a drug-resistant cell clone (100×) and ZYM-SIEC02 cells at passage 90 (100×).No obvious morphological differences were observed between ZYM-SIEC02 cells and primary SIECs. B. Electron micrographs of monolayer cultures of ZYM-SIEC02 cells indicate microvilli (MV) in 3 dimensions andin monolayer culture; tight junctions (TJ) and a small intestine glandular configuration (SIGC) (Red arrow).
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pone-0110916-g001: Morphological features of pSIECs and ZYM-SIEC02 cells.A. Cellular morphology of pSIECs at 7 days in culture (100×) and at passage 5 (100×); a drug-resistant cell clone (100×) and ZYM-SIEC02 cells at passage 90 (100×).No obvious morphological differences were observed between ZYM-SIEC02 cells and primary SIECs. B. Electron micrographs of monolayer cultures of ZYM-SIEC02 cells indicate microvilli (MV) in 3 dimensions andin monolayer culture; tight junctions (TJ) and a small intestine glandular configuration (SIGC) (Red arrow).

Mentions: Cell material obtained from porcine mid-jejunum consisted of cell aggregates or organoids that adhered to collagen-coated culture flasks and formed circular proliferating foci (Fig. 1A, upper left). Initial expansion of the foci resulted in fusion of the cell plaques, forming a confluent cell layer within 10–12 days due to the presence of residual multicellular organoids dispersed in the newly formed monolayer. During the first passage, epithelial cell sheets exhibited a homogeneous cobblestone-like morphology. No obvious morphological changes were observed during prolonged passage in culture, defined as 8–10 passages at a split ratio of 1∶2 over 4–5 weeks (Fig. 1A, upright). Cells reached confluence within 2–3 days during the early passages (p1–p6). However, a progressive slowdown in the proliferation rate was observed until cells reached senescence. Eleven passages later, cells became enlarged and the cobblestone-like morphology was lost, even though the expression of cytokeratin 18 was maintained (data not shown).


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)

Morphological features of pSIECs and ZYM-SIEC02 cells.A. Cellular morphology of pSIECs at 7 days in culture (100×) and at passage 5 (100×); a drug-resistant cell clone (100×) and ZYM-SIEC02 cells at passage 90 (100×).No obvious morphological differences were observed between ZYM-SIEC02 cells and primary SIECs. B. Electron micrographs of monolayer cultures of ZYM-SIEC02 cells indicate microvilli (MV) in 3 dimensions andin monolayer culture; tight junctions (TJ) and a small intestine glandular configuration (SIGC) (Red arrow).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110916-g001: Morphological features of pSIECs and ZYM-SIEC02 cells.A. Cellular morphology of pSIECs at 7 days in culture (100×) and at passage 5 (100×); a drug-resistant cell clone (100×) and ZYM-SIEC02 cells at passage 90 (100×).No obvious morphological differences were observed between ZYM-SIEC02 cells and primary SIECs. B. Electron micrographs of monolayer cultures of ZYM-SIEC02 cells indicate microvilli (MV) in 3 dimensions andin monolayer culture; tight junctions (TJ) and a small intestine glandular configuration (SIGC) (Red arrow).
Mentions: Cell material obtained from porcine mid-jejunum consisted of cell aggregates or organoids that adhered to collagen-coated culture flasks and formed circular proliferating foci (Fig. 1A, upper left). Initial expansion of the foci resulted in fusion of the cell plaques, forming a confluent cell layer within 10–12 days due to the presence of residual multicellular organoids dispersed in the newly formed monolayer. During the first passage, epithelial cell sheets exhibited a homogeneous cobblestone-like morphology. No obvious morphological changes were observed during prolonged passage in culture, defined as 8–10 passages at a split ratio of 1∶2 over 4–5 weeks (Fig. 1A, upright). Cells reached confluence within 2–3 days during the early passages (p1–p6). However, a progressive slowdown in the proliferation rate was observed until cells reached senescence. Eleven passages later, cells became enlarged and the cobblestone-like morphology was lost, even though the expression of cytokeratin 18 was maintained (data not shown).

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