Liebe Heather, Schlegel Camille, Cai Xue, Golubkova Alena, Leiva Tyler, Berry William L, Hunter Catherine J
Division of Pediatric Surgery, Oklahoma Children's Hospital.
Oklahoma University Health Sciences Center.
J Vis Exp. 2022 Jul 27(185). doi: 10.3791/64215.
Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein β-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.
肠类器官是坏死性小肠结肠炎（NEC）等炎症性肠病研究中一种新兴的研究工具。传统上，它们以基底外侧向外（BO）的构象生长，上皮细胞的顶端表面面向内腔。在这个模型中，进入肠类器官的腔表面进行治疗和实验具有挑战性，这限制了研究宿主-病原体相互作用的能力。为了规避这一问题，创建了一种用于坏死性小肠结肠炎的新生儿顶端向外（AO）模型。由于肠上皮细胞通透性变化是NEC的特征性表现，本方案概述了使用荧光素黄（LY）作为细胞旁通透性的标志物。LY通过所有三种主要的细胞旁途径穿过肠上皮屏障：孔隙、渗漏和无限制途径。在AO模型中使用LY可以更广泛地研究NEC中的通透性。经机构审查委员会（IRB）批准并获得家长同意后，从人类早产新生儿中收集肠道组织的手术样本。通过隐窝分离收获肠道干细胞并用于培养肠类器官。将肠类器官培养至成熟，然后转化为AO构象或保持BO构象。这些肠类器官要么不进行处理（对照），要么用脂多糖（LPS）处理并置于缺氧条件下以诱导体外NEC。使用LY评估通透性。顶端蛋白闭合蛋白-1和基底外侧蛋白β-连环蛋白的免疫荧光染色证实了AO构象。与对照相比，用LPS和缺氧处理的AO和BO肠类器官均显示细胞旁通透性显著增加。与对照相比，AO和BO肠类器官均显示处理后的肠类器官内腔对LY的摄取增加。在AO肠类器官模型中使用LY可以研究细胞旁通透性的所有三种主要途径。与BO肠类器官模型相比，它还可以研究宿主-病原体相互作用以及这可能如何影响通透性。
