Overexpression of the Apical Sodium-Dependent Bile Acid Transporter to Replicate Necrotizing Enterocolitis in IEC-6 Cells

Persistent Link:
http://hdl.handle.net/10150/271218
Title:
Overexpression of the Apical Sodium-Dependent Bile Acid Transporter to Replicate Necrotizing Enterocolitis in IEC-6 Cells
Author:
Rogan, Daniel Thomas
Issue Date:
May-2012
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:
text; Electronic Thesis
Degree Name:
B.S.
Degree Level:
bachelors
Degree Program:
Honors College; Physiology
Degree Grantor:
University of Arizona

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleOverexpression of the Apical Sodium-Dependent Bile Acid Transporter to Replicate Necrotizing Enterocolitis in IEC-6 Cellsen_US
dc.creatorRogan, Daniel Thomasen_US
dc.contributor.authorRogan, Daniel Thomasen_US
dc.date.issued2012-05-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.abstractThis 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.en_US
dc.typetexten_US
dc.typeElectronic Thesisen_US
thesis.degree.nameB.S.en_US
thesis.degree.levelbachelorsen_US
thesis.degree.disciplineHonors Collegeen_US
thesis.degree.disciplinePhysiologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
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