IPEC-J2 cells treated with cholera toxin for 8h. Sus scrofa

The molecular mechanisms involved in host pathogen interactions at mucosal surfaces needs to be better understood in order to develop immune-mediated methods of protection against pathogens. Cholera toxin (CT) has the rare ability for a protein of inducing robust mucosal immunity in the gut and is therefore an excellent model with which to determine mechanisms of adjuvanticity and immunogenicity at intestinal mucosal surfaces. Jejunal epithelial cells are one of the first sites of antigen encounter. Therefore a porcine intestinal epithelial cell line, IPEC-J2, was cultured in 6-well transwell plates in the presence or absence of 50 ng/ml cholera toxin for up to 8 hours and the cell layer was harvested for gene expression analysis using the Affymetrix porcine genome array and real-time PCR analysis. Affymetrix analysis identified, and real-time PCR analysis of 15 genes confirmed, an increase in gene expression for 59 genes and a decrease in gene expression for 14 genes under CT treatment. An 8 hour time course of expression revealed that by 2-4 hours after CT treatment, all 10 upregulated genes were differentially expressed and by 4-6 hours after CT treatment 3 of the 5 downregulated genes were differentially expressed. These data suggest that the potent mucosal adjuvanticity and immunogenicity of CT derives from rapid alterations in gene expression at the site of first antigen encounter with the immune system. Characterization of early immune gene expression may elucidate potential biological mechanisms for mucosal immune induction leading to the development of effective vaccines against enteric pathogens. Keywords: Treatment comparison, cholera toxin vs untreated at 8h time point Overall design: Confluent IPEC-J2 cell monolayers were treated with or without 50 ng/ml cholera toxin in transwell dishes for 8h. The experiment was repeated 3 times for a total of 6 chips, 3 cholera toxin treated and 3 untreated. Gene expression was compared between cholera toxin treated and untreated cells.

为开发针对病原体的免疫介导防御策略，我们亟需更深入地阐明黏膜表面宿主-病原体互作的分子机制。霍乱毒素（cholera toxin, CT）作为一种蛋白质，具备诱导肠道强效黏膜免疫的罕见特性，因此是研究肠黏膜表面佐剂活性与免疫原性机制的极佳模型。空肠上皮细胞是抗原首次接触的位点之一。为此，研究人员将猪源肠道上皮细胞系IPEC-J2接种于6孔Transwell板中，分别添加或不添加50 ng/ml的霍乱毒素，培养时长最长达8小时，随后收集细胞层，采用Affymetrix猪基因组表达芯片（Affymetrix porcine genome array）与实时荧光定量PCR（real-time PCR）进行基因表达分析。Affymetrix芯片分析共筛选出差异表达基因，经15个基因的实时荧光定量PCR验证，霍乱毒素处理组共有59个基因表达上调、14个基因表达下调。8小时表达时间进程分析显示，霍乱毒素处理后2-4小时，全部10个上调基因均已呈现差异表达；处理后4-6小时，5个下调基因中的3个已出现差异表达。上述数据表明，霍乱毒素的强效黏膜佐剂活性与免疫原性，源于其在免疫系统首次接触抗原的位点处快速诱导基因表达改变。对早期免疫基因表达特征的解析，或可阐明黏膜免疫诱导的潜在生物学机制，进而助力开发针对肠道病原体的高效疫苗。关键词：处理组对照，8小时时间点霍乱毒素处理组与未处理组对比。实验设计：将汇合状态的IPEC-J2细胞单层置于Transwell培养皿中，分别添加或不添加50 ng/ml的霍乱毒素，培养8小时。实验重复3次，共计制备6张芯片，其中霍乱毒素处理组与未处理组各3张。本研究对两组细胞的基因表达水平进行了对比分析。