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

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-? 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. TNF? mRNA levels were significantly decreased after LPS stimulation and TNF-? 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.

SUBMITTER: Wang J

PROVIDER: S-EPMC4206455 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Wang Jing J Hu Guangdong G Lin Zhi Z He Lei L Xu Lei L Zhang Yanming Y

PloS one 20141022 10

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 ...[more]

PMID: 25337908

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Project description:Food and feed supplements containing microorganisms with probiotic potential are of increasing interest due to their healthy promoting effect on human and animals. Their mechanism of action is still unknown. Using a microarray approach, the aim of this study was to investigate the differences in genome-wide gene expression induced by a mixture of three Lactobacillus strains (L. rhamnosus, L. plantarum, and L. paracasei) in intestinal porcine epithelial cells (IPEC-1) and to identify the genes and pathways involved in intestinal barrier functions. Undifferentiated IPEC-1 cells seeded at a density of 2.0 × 10⁵/mL in 24-wells culture plates were cultivated at 37 °C and 5% CO₂ until they reached confluence (2⁻3 days). Confluent cells monolayer were then cultivated with 1 mL of fresh lactobacilli (LB) mixture suspension prepared for a concentration of approximately 3.3 × 10⁷ CFU/mL for each strain (1 × 10⁸ CFU/mL in total) for 3 h and analyzed by microarray using Gene Spring GX v.11.5. The functional analysis showed that 1811 of the genes modulated by LB treatment are involved in signaling (95% up-regulation, 121 genes with a fold change higher than 10). The most enhanced expression was registered for AXIN2 (axis inhibition protein 2-AXIN2) gene (13.93 Fc, p = 0.043), a negative regulator of β-catenin with a key role in human cancer. LB affected the cellular proliferation by increasing 10 times (Fc) the NF1 gene encoding for the neurofibromin protein, a tumor suppressor that prevent cells from uncontrolled proliferation. The induction of genes like serpin peptidase inhibitor, clade A member 3 (SERPINA 3), interleukin-20 (IL-20), oncostatin M(OSM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the suppression of chemokine (C-X-C motif) ligand 2/macrophage inflammatory protein 2-alpha (CXCL-2/MIP-2), regulator of G-protein signaling 2 (RGS2), and of pro-inflammatory interleukin-18 (IL-18) genes highlights the protective role of lactobacilli in epithelial barrier function against inflammation and in the activation of immune response. Gene overexpression was the predominant effect produced by lactobacilli treatment in IPEC-1 cells, genes related to signaling pathways being the most affected. The protective role of lactobacilli in epithelial barrier function against inflammation and in the activation of immune response was also noticed.

Project description:BackgroundMucosal melanoma has characteristically distinct genetic features and typically poor prognosis. The lack of representative mucosal melanoma models, especially cell lines, has hindered translational research on this melanoma subtype. In this study, we aimed to establish and provide the biological properties, genomic features and the pharmacological profiles of a mucosal melanoma cell line that would contribute to the understanding and treatment optimization of molecularly-defined mucosal melanoma subtype.MethodsThe sample was collected from a 67-year-old mucosal melanoma patient and processed into pieces for the establishment of cell line and patient-derived xenograft (PDX) model. The proliferation and tumorigenic property of cancer cells from different passages were evaluated, and whole-genome sequencing (WGS) was performed on the original tumor, PDX, established cell line, and the matched blood to confirm the establishment and define the genomic features of this cell line. AmpliconArchitect was conducted to depict the architecture of amplified regions detected by WGS. High-throughput drug screening (HTDS) assay including a total of 103 therapeutic agents was implemented on the established cell line, and selected candidate agents were validated in the corresponding PDX model.ResultsA mucosal melanoma cell line, MM9H-1, was established which exhibited robust proliferation and tumorigenicity after more than 100 serial passages. Genomic analysis of MM9H-1, corresponding PDX, and the original tumor showed genetic fidelity across genomes, and MM9H-1 was defined as a triple wild-type (TWT) melanoma subtype lacking well-characterized "driver mutations". Instead, the amplification of several oncogenes, telomerase reverse transcriptase (TERT), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), melanocyte Inducing transcription factor (MITF) and INO80 complex ATPase subunit (INO80), via large-scale genomic rearrangement potentially contributed to oncogenesis of MM9H-1. Moreover, HTDS identified proteasome inhibitors, especially bortezomib, as promising therapeutic candidates for MM9H-1, which was verified in the corresponding PDX model in vivo.ConclusionsWe established and characterized a new mucosal melanoma cell line, MM9H-1, and defined this cell line as a TWT melanoma subtype lacking well-characterized "driver mutations". The MM9H-1 cell line could be adopted as a unique model for the preclinical investigation of mucosal melanoma.

Dataset Information

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

Publications

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

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