Biological processes induced by ZnO, Amoxicillin, and Rye in cultured Intestinal Porcine Epithelial Cells (IPEC-J2)

The objective of this study is to investigate to what extent gene expression data of dietary interventions generated in IPEC-J2 in vitro model overlap with in vivo data. Gene expression was recorded in IPEC-J2 cells upon exposure to three different dietary interventions commonly used in livestock. In a further step, we compared the results with mucosal gene expression responses, as measured in animals exposed to the same compounds via the diet. As compounds we used zinc oxide, rye and the antibiotic amoxicillin. The GEO accession numbers of the in vivo datasets are provided in the paper "Enrichment of in vivo determined transcription data from dietary intervention studies with in vitro data provides improved insight into gene regulation mechanisms" (submitted to "Genes and Nutrition"). IPEC-J2 cells were grown for 7 days at 37 ºC and 5% CO2 using 1:1 DMEM/Ham’s F10 1:1 medium supplemented with 5% FCS without antibiotics. For all tests, confluent monolayers were washed twice with medium without FCS (hereafter denoted as medium) and incubated for 1 hour with this medium. Hereafter, the medium was discarded and an additive dissolved/suspended in medium was added. Concentrations of 2.5% w/v Amoxicillin (brand name Octacillin), 0.03125% w/v of ZnO, and of a 3-fold diluted suspension of the diets containing 0%, 5% or 10% w/v Rye in culture medium were used for incubation of IPEC-J2 monolayers for a period of 2 and 6h. All incubations were tested in duplicate and for each type of additive duplicate control wells containing no additive (only culture medium) were incubated for 2 and 6h. After incubation total RNA was extracted. Each duplicate incubation was hybridized separately on a microarray patch. IPEC-J2 enterocyte cell line derived from the jejunum of piglets, host-feed interaction

本研究旨在探究IPEC-J2体外模型中获取的膳食干预基因表达数据与体内数据的重叠程度。研究针对家畜常用的三种不同膳食干预方式处理IPEC-J2细胞，记录其基因表达情况；随后将所得结果与通过膳食接触相同化合物的动物的黏膜基因表达响应进行对比。本次研究使用的受试化合物为氧化锌、黑麦以及抗生素阿莫西林。体内数据集的GEO基因表达综合数据库（Gene Expression Omnibus，GEO）登录号已发表于论文《通过体外数据富集膳食干预研究的体内转录组数据以深入解析基因调控机制》（已提交至"Genes and Nutrition"）。IPEC-J2细胞以DMEM与Ham’s F10按1:1体积比混合的培养基进行培养，该培养基添加5%胎牛血清（Fetal Calf Serum，FCS）且不含抗生素，培养条件为37℃、5%CO₂，培养时长7天。所有实验中，汇合单层细胞先用无FCS的培养基（后文简称基础培养基）洗涤两次，随后以该基础培养基孵育1小时；之后弃去基础培养基，加入溶解或悬浮于基础培养基中的受试物。孵育IPEC-J2单层细胞的受试物浓度设置为：2.5%重量体积比（weight/volume，w/v）阿莫西林（商品名Octacillin）、0.03125%重量体积比（w/v）氧化锌，以及3倍稀释的、添加有0%、5%或10%重量体积比黑麦的日粮悬浮液，孵育时长分别为2小时和6小时。所有孵育实验均设置重复孔，同时设置不含受试物的对照孔（仅添加基础培养基），对照孔同样设置重复并分别孵育2小时和6小时。孵育结束后提取总RNA，每份重复孵育样本均单独在一个微阵列芯片点样单元进行杂交。IPEC-J2是源自仔猪空肠的肠上皮细胞系，用于宿主-饲料互作研究。