Abstract

Porcine gastric epithelial cells (PGECs) serve as a valuable model for studying the molecular and pathogenic mechanisms of the stomach. However, PGECs face limitations such as isolation challenges, short lifespan, and restricted proliferation. To address this, we established an immortalized PGECs (i-PGECs) to enable in vitro investigation of pathogen infection mechanisms. Primary PGECs were isolated from the acid-secreting glands using stepwise digestion with multiple enzymes (dispase II/collagenase I/hyaluronidase). Immortalization was achieved via lentiviral vectors expressing simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase ( hTERT ), with successful expression confirmed by qRT-PCR (P < 0.05). Epithelial identity of i-PGECs was confirmed by stable expression of CK18, EpCAM, and E-cadherin, as shown by qRT-PCR and immunofluorescence. i-PGECs retained the morphological and ultrastructural features of PGECs and exhibited enhanced proliferation, as demonstrated by WST-8 assays, apoptosis and cell cycle analysis, karyotyping, and transmission electron microscopy (TEM). Telomere length analysis and scratch wound assays demonstrated stable telomere maintenance and consistent migration capacity unaffected by passaging. RNA-sequencing and differential expressed genes (DEGs) analysis revealed significantly upregulating of genes involved in cell proliferation pathways ( P  < 0.01). Following aflatoxin B1 (AFB1) exposure, i-PGECs significantly upregulated immune-related factors, such as NPC1 and PLAUR ( P  < 0.01). CRISPR/Cas9-mediated knockout of NPC1 in i-PGECs conferred increased resistance to AFB1-induced cytotoxicity, as shown by WST-8 assay. The i-PGECs remained stable after more than 50 passages, supporting their use as a reliable for in vitro model investigating the mechanisms of toxicity infection in the porcine gastric epithelium.