OVEREXPRESSION OF THE APICAL SODIUM-DEPENDENT BILE ACID TRANSPORTER TO REPLICATE NECROTIZING ENTEROCOLITIS IN CACO-2 CELLS

Kevin Mistler , Melissa D. Halpern, Teresa Estrada

OVEREXPRESSION OF THE APICAL SODIUM-DEPENDENT BILE ACID TRANSPORTER TO REPLICATE NECROTIZING ENTEROCOLITIS IN CACO-2 CELLS

Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal diseases in premature infants. It involves necrosis of intestinal tissue in the ileum and colon and is potentially life threatening. Although the pathophysiology of NEC is poorly understood, disease development is associated with an accumulation of ileal bile acids. The apical sodium-dependent bile acid transporter (ASBT) transports luminal bile acids into enterocytes. ASBT is up-regulated in NEC, and this increase results in greater intracellular bile acid levels, which exacerbates the ileal inflammation observed in NEC. The purpose of this study is to test a method of overexpressing ASBT in CaCo-2 cells to replicate what occurs in NEC. The expression vector pcDNA4/TO was modified to pDR1019, containing the Rattus norvegicus ASBT coding sequence. CaCo-2 cells will be transfected with varying ratios of pDR1019 to pcDNA6/TR, a tetracycline-controlled repression vector. After selection for cells containing both plasmids, RNA extraction and qrt-PCR will be performed to determine the optimal ratio for ASBT overexpression. Subsequent transfections will be performed using the appropriate pDR1019:pcDNA6/TR ratio, and following transfection, cells will undergo treatments with chenodeoxycholic acid (CDCA), tumor necrosis factor-alpha (TNF-α), and interleukin 18 (IL18) to investigate the effects of bile acids and pro-inflammatory cytokines on ASBT when it is overexpressed.

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