Overexpression of the Apical Sodium-Dependent Bile Acid Transporter to Replicate Necrotizing Enterocolitis in IEC-6 Cells
Publisher
The University of Arizona.Rights
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.Abstract
This study tested a method of overexpressing the apical sodium-dependent bile acid transporter (ASBT) in IEC-6 cells to replicate necrotizing enterocolitis (NEC), a gastrointestinal disease prevalent in premature infants. The expression vector pcDNA4/TO was altered to pDR1019, containing the Rattus norvegicus ASBT coding sequence. Sequencing confirmed the ASBT sequence in pDR1019 using forward, reverse, and midsequence primers (respective E-values: 0.0, 0.0, and 2e ⁻¹⁷⁴). IEC-6 cells were transfected with varying ratios of pcDNA6/TR (tetracycline-controlled repression vector) and pDR1019: 6:1, 15:1, and 30:1 pcDNA6/TR:pDR1019. Using relative quantitative real-time PCR (qrt-PCR), the 30:1 transfection had the greatest fold-change difference of ASBT mRNA expression relative to non-transfected IEC-6 cells (overexpression-induced and noninduced trials: 3120.0-fold and 1445.6-fold, respectively). To test the effects of bile acids and cytokines on ASBT expression in IEC-6 cells with overexpressed ASBT, a 30:1 pcDNA6/TR:pDR1019 transfection was performed, followed by treatments of chenodeoxycholic acid (CDCA), tumor necrosis factor-alpha (TNF-α), and interleukin 18 (IL18). According to a qrt-PCR, the ASBT mRNA expression fold-change of non-transfected trials were: CDCA (2.09x10⁹-fold), TNF-α (0.39-fold), IL18 (2.12x10⁹-fold); insufficient cells survived the transfection followed by treatments to yield usable RNA. Using this cell-based model to replicate NEC will aid future molecularly-based investigations of the disease.Type
textElectronic Thesis
Degree Name
B.S.Degree Level
bachelorsDegree Program
Honors CollegePhysiology