|GUPTA, ANAMIKA - University Of Arkansas
|LIYANAGE, ROHANA - University Of Arkansas
|Donoghue, Ann - Annie
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/13/2017
Publication Date: 11/13/2018
Citation: Gupta, A., Liyanage, R., Donoghue, A.M. 2018. Development of a chicken enterocyte culture to study its functional physiology [abstract]. p. 25.
Technical Abstract: We developed a method to culture chicken intestinal enterocytes, the cells that absorb and form protective barriers against enteric bacteria, to study their functional physiologies. Using intestinal villi, harvested from day old broiler chicks, the enterocytes were isolated by sequential digestion with streptococcal hyaluronidase and trypsin followed by a density gradient centrifugation over Histopaque. The isolated clusters of cells were plated in Dulbecco’s modified Eagle’s medium (DMEM) containing antibiotic/antimycotic, fetal bovine serum, epithelial cell growth factor, and insulin transferrin selenium (ITS) supplements, which allowed the epithelial-like cells to grow. These cells were subcultured for 2 passages upon which they were plated at a concentration of 105 cells/well in 6 well plates to semi-confluent. We tested the effect of butyrate, a short chain fatty acid, known to be beneficial for intestinal health using proteomic approach, to find its regulatory effects. Quadruplicate cultures of both control and butyrate treated cells were lysed and the soluble proteins in the lysates were subjected to electrophoresis on a 4-20% gradient gel, stained, and each lane was excised horizontally into 2 segments. The gel segments were then subjected to reduction/alkylation followed by digestion with trypsin. The tryptic peptides were then analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) to identify and quantify proteins in the sample. The differentially regulated proteins were identified using Scaffold software. We identified 487 proteins, out of which 16 were uniquely expressed in control enterocytes, 8 in Butyrate treated cells and 404 found in both. Of 487 proteins, 104 proteins were downregulated and 10 proteins are upregulated by butyrate. The proteins that are regulated by butyrate are Annexin, Cadherin13, Histone H2A. Our study concluded that butyrate may have impact on cell motility, cytoskeleton changes and energy metabolism.