Date of Graduation

12-2014

Document Type

Thesis

Degree Name

Master of Science in Animal Science (MS)

Degree Level

Graduate

Department

Animal Science

Advisor/Mentor

Maxwell, Charles V.

Committee Member

Rosenkrans, Charles F. Jr.

Second Committee Member

Kegley, Elizabeth B.

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.

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