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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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Growth curves, apoptosis and cell cycle analysis of SIECs before and after hTERT transfection.A. Growth of pSIECs and ZYM-SIEC02 cells. Data are represented as the mean ± SD of 3 independent experiments. B. Apoptosis analysis of control pSIECs and ZYM-SIEC02 cells, The percentage of apoptosis in pSIECs and ZYM-SIEC02 cells under basal growth conditions was 26.9% and 11.4% (Q2+Q4), respectively. C. Cell cycle distributions of control SIECs and ZYM-SIEC02 cells, the percentages of pSIECs and ZYM-SIEC02 cells in S phase were 20.66% and 31.65%, respectively.
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pone-0110916-g003: Growth curves, apoptosis and cell cycle analysis of SIECs before and after hTERT transfection.A. Growth of pSIECs and ZYM-SIEC02 cells. Data are represented as the mean ± SD of 3 independent experiments. B. Apoptosis analysis of control pSIECs and ZYM-SIEC02 cells, The percentage of apoptosis in pSIECs and ZYM-SIEC02 cells under basal growth conditions was 26.9% and 11.4% (Q2+Q4), respectively. C. Cell cycle distributions of control SIECs and ZYM-SIEC02 cells, the percentages of pSIECs and ZYM-SIEC02 cells in S phase were 20.66% and 31.65%, respectively.

Mentions: The growth curves of ZYM-SIEC02 cells and control pSIEC are shown in Figure 3A. In order to ascertain whether hTERT expression affected cell proliferation or cell cycle progression, cell cycle distribution and basal levels of apoptosis in pSIECs and ZYM-SIEC02 cells were determined by flow cytometry. The percentages of apoptotic cells in pSIECs and ZYM-SIEC02 cultures were 26.9% and 11.4% (Q2+Q4), respectively (Fig. 3B). The percentages of pSIECs and ZYM-SIEC02 cells in S phase were 20.7% and 31.7%, respectively (Fig. 3C), indicating that the immortalized ZYM-SIEC02 cell line exhibits increased proliferative activity and a lower incidence of apoptosis compared to that of primary swine intestinal epithelial cells.


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)

Growth curves, apoptosis and cell cycle analysis of SIECs before and after hTERT transfection.A. Growth of pSIECs and ZYM-SIEC02 cells. Data are represented as the mean ± SD of 3 independent experiments. B. Apoptosis analysis of control pSIECs and ZYM-SIEC02 cells, The percentage of apoptosis in pSIECs and ZYM-SIEC02 cells under basal growth conditions was 26.9% and 11.4% (Q2+Q4), respectively. C. Cell cycle distributions of control SIECs and ZYM-SIEC02 cells, the percentages of pSIECs and ZYM-SIEC02 cells in S phase were 20.66% and 31.65%, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110916-g003: Growth curves, apoptosis and cell cycle analysis of SIECs before and after hTERT transfection.A. Growth of pSIECs and ZYM-SIEC02 cells. Data are represented as the mean ± SD of 3 independent experiments. B. Apoptosis analysis of control pSIECs and ZYM-SIEC02 cells, The percentage of apoptosis in pSIECs and ZYM-SIEC02 cells under basal growth conditions was 26.9% and 11.4% (Q2+Q4), respectively. C. Cell cycle distributions of control SIECs and ZYM-SIEC02 cells, the percentages of pSIECs and ZYM-SIEC02 cells in S phase were 20.66% and 31.65%, respectively.
Mentions: The growth curves of ZYM-SIEC02 cells and control pSIEC are shown in Figure 3A. In order to ascertain whether hTERT expression affected cell proliferation or cell cycle progression, cell cycle distribution and basal levels of apoptosis in pSIECs and ZYM-SIEC02 cells were determined by flow cytometry. The percentages of apoptotic cells in pSIECs and ZYM-SIEC02 cultures were 26.9% and 11.4% (Q2+Q4), respectively (Fig. 3B). The percentages of pSIECs and ZYM-SIEC02 cells in S phase were 20.7% and 31.7%, respectively (Fig. 3C), indicating that the immortalized ZYM-SIEC02 cell line exhibits increased proliferative activity and a lower incidence of apoptosis compared to that of primary swine intestinal epithelial cells.

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