Global transcriptional response of porcine intestinal epithelial cell lines to Salmonella enterica serovar Typhimurium and Choleraesuis: IPEC-J2 infected with S. choleraesuis. Sus scrofa

The GeneChip Porcine Genome Array was used to identify the transcriptional response upon either Salmonella typhimurium (ST) or Salmonella choleraesuis (SC) infection in two porcine epithelial cell lines (IPEC-J2, from jejunum and IPI-2I, from ileum) during 2 and 4 hours post infection. The objectives in this study were first, to identify the different response between the epithelial cell lines from different gut regions; second, to study how the Salmonella serotypes used could elicit a different host response; and third, to determine the effect of the time-points on the differentially gene expression. Overall design: Epithelial cells were seeded into 6-well tissue culture plates and grown to confluence in 5% CO2 at 37ºC. Monolayers were infected for 1 h. with Salmonella typhimurium or Salmonella choleraesuis serotypes (MOI 1:10) or incubated with media alone (Control cells). Extracellular bacteria were removed, and cultures were further incubated during 2 and 4 h. in the presence of 50 µg/ml of the non-cell-permeant antibiotic gentamicin to kill remaining extracellular bacteria. The experiment was developed in triplicate in order to ensure a proper robustness in the microarray statistical analysis. An indirect ELISA was used to confirm cell activation by means of IL8 detection in cell culture medium. After each time-point, cells were lysed and RNA extracted.

本研究使用基因芯片猪基因组阵列（GeneChip Porcine Genome Array），旨在鉴定两种猪上皮细胞系——来源于空肠的IPEC-J2与来源于回肠的IPI-2I——在感染鼠伤寒沙门氏菌（Salmonella typhimurium，ST）或猪霍乱沙门氏菌（Salmonella choleraesuis，SC）后，于感染后2小时与4小时的转录应答情况。本研究的目标分为三点：其一，鉴定不同肠道区域来源的上皮细胞系之间的转录应答差异；其二，探究不同沙门氏菌血清型如何诱导宿主产生不同的应答反应；其三，明确时间点对差异基因表达的影响。总体实验设计如下：将上皮细胞接种至6孔组织培养板，于37℃、5%CO₂培养环境中培养至细胞汇合。单层细胞分别以感染复数（MOI）1:10的鼠伤寒沙门氏菌、猪霍乱沙门氏菌血清型感染1小时，或仅用培养基孵育（设置对照组细胞）。移除胞外细菌后，向培养体系中加入50μg/ml的非细胞渗透性抗生素庆大霉素，继续孵育2小时与4小时以杀灭残留的胞外细菌。本实验设置三次生物学重复，以保障微阵列统计分析的稳健性。采用间接ELISA法检测细胞培养液中的IL8，以验证细胞是否被激活。在每个时间点收集细胞并裂解，提取总RNA。