Date of Graduation

12-2014

Document Type

Thesis

Degree Name

Master of Science in Animal Science (MS)

Degree Level

Graduate

Department

Animal Science

Advisor

Charles Maxwell

Committee Member

Charles Rosenkrans

Second Committee Member

Beth Kegley

Keywords

ART-PCR, Growth Rate, Immnue Cells Gene Expression, Innate Immune, IPEC-J2, LPS Challenge

Abstract

The objective of this study was to evaluate the intestinal porcine epithelial cell-jejunum 2 (IPEC-J2) cell line as a model to study the innate immune function of live pigs. Growth rates of IPEC-J2 cells in T-75 flasks and 96-well plates were evaluated using a hemocytometer and spectrophotometer for cell quantity measurements to determine growth rate and doubling time. Growth rates of IPEC-J2 cells in T-75 flasks and 96-well plates were 0.4016 and 0.2851 times of doubling/day respectively with a doubling time of 1.73 d and 2.43 d, respectively. Confluent IPEC-J2 monolayers were tested at five time intervals (0, 1, 2, 4, and 6 h) and four LPS concentrations (0, 0.1, 1, and 10 μg/mL). Under these treatments, relative gene expression of GM-CSF, IL-1β, TNF-α, IL-6, IL-8, IL-10, TLR1, TLR2, TLR3, TLR4, TLR6, TLR8 and TLR10 were evaluated by quantitative RT-PCR. There were no LPS concentration × culture time interactions observed for any gene (P > 0.13). Main effects were analyzed. The GM-CSF, IL-8, and TLR4 were significantly stimulated by LPS challenge at increasing culture time and the expression peaked at 2, 4, and 4 h respectively (P < 0.05). Expression of TNF-α tended to increase linearly with increasing LPS concentration and decrease linearly with increasing culture time (P = 0.10). TLR2 tended to be up regulated by increasing LPS challenge time (Quadratic effect, P = 0.08). Increasing culture time lead to a down regulation of IL-6 expression, and reached a low point at 4 h (Quadratic effect, P < 0.05). TLR3 tended to be down regulated with increasing culture time (Quadratic effect, P = 0.10). Results of the current study suggest that the IPEC-J2 cell line can be used as a model for evaluating the impact of specific bacteria on immune response in vitro.