Stewart Amy Stieler, Kopper Jamie J, McKinney-Aguirre Caroline, Veerasamy Brittany, Sahoo Dipak Kumar, Freund John M, Gonzalez Liara M
Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, United States.
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA, United States.
Front Vet Sci. 2024 Oct 22;11:1455262. doi: 10.3389/fvets.2024.1455262. eCollection 2024.
Gastrointestinal disease is a leading cause of death in mature horses. A lack of modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5-7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.
胃肠道疾病是成年马匹死亡的主要原因。缺乏相关模型阻碍了新型疗法的开发。本研究的目的是开发并进一步表征源自马空肠的小肠单层细胞培养物，包括确定肠道通透性和修复的正常测量值。利用从马空肠的尸检样本中获得的三维肠类器官来培养汇合的上皮单层。使用组织学、逆转录聚合酶链反应（RT-PCR）、RNAscope、蛋白质免疫荧光和透射电子显微镜确认了分化的肠道上皮细胞类型和紧密连接的存在。评估跨上皮电阻（TER）和大分子通量作为细胞旁和跨细胞通透性的测量指标。利用划痕试验来模拟和评估肠道修复。单层细胞培养物在约5-7天达到100%汇合。马空肠单层被确认为起源于上皮细胞，鉴定出分化的肠道上皮细胞类型并发现紧密连接蛋白的证据。肠道屏障的功能通过获得生理正常的TER值（179.9±33.7欧姆*厘米）和有限的大分子通量（60分钟时为22±8.8%）得到支持。此外，在划痕损伤后，上皮细胞单层在24小时内迁移以闭合间隙缺损。总之，本研究描述了一种用于马空肠的新型肠道上皮单层细胞培养物的开发，并提供了肠道上皮细胞分化、形成生理相关屏障功能以及用作肠道修复模型以测试马绞痛潜在疗法的证据。
