Apical-Out Enteroids as an Innovative Model for Necrotizing Enterocolitis.
Apical-Out Enteroids as an Innovative Model for Necrotizing Enterocolitis.
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DOI：
10.1016/j.jss.2022.11.048
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发表时间：
2023-03
期刊：
影响因子：
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通讯作者：
Heather L. Liebe;Camille Schlegel;Xue Cai;A. Golubkova;Christopher Loerke;Tyler Leiva;C. Hunter
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文献类型：
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作者：
Heather L. Liebe;Camille Schlegel;Xue Cai;A. Golubkova;Christopher Loerke;Tyler Leiva;C. Hunter
前言坏死性小肠结肠炎（NEC）是早产儿常见的消化道疾病。我们以前验证了NEC肠模型来自人类婴儿肠组织。典型的肠样结构是基底外侧向外（BO），没有直接进入管腔（顶端）表面。心尖入路是必要的，以允许病原体与肠上皮屏障相互作用的生理比较。我们假设，顶端出（AO）肠将提供一个相关的NEC模型来研究这种relationship. MethodsAfter机构审查委员会批准（#11610-11611），新生儿肠组织收集手术标本。收集干细胞;产生肠样细胞并以BO构象生长至成熟，然后翻转至AO。未处理或用100 μg/mL脂多糖和缺氧处理24小时的肠状体。蛋白和基因表达进行了分析炎症标志物，紧密连接（TJ）蛋白和渗透性NEC.Resultsapical TJ蛋白zonula occludens-1和基底外侧蛋白β-catenin免疫荧光证实AO配置的特点。与对照组相比，AO处理的肠样组织中肿瘤坏死因子-α的信使RNA（P= 0.001）和蛋白水平（P< 0.0001）显著增加。与对照组相比，经治疗的AO类肠细胞中Toll样受体4的校正总细胞荧光显著增加（P= 0.002）。在治疗的AO肠样组织中，发现Occludin具有显著降低的信使RNA（P= 0.003）。其他TJ蛋白claudins-1、claudins-4和闭合小带-1的表达在治疗的AO类肠中显著降低（P< 0.05）。该模型允许NEC中病原体和肠上皮屏障之间的相互作用的生物学相关的调查。
IntroductionNecrotizing enterocolitis (NEC) is a gastrointestinal disease of premature neonates. We previously validated a NEC enteroid model derived from human infant intestinal tissue. Typical enteroid configuration is basolateral-out (BO) without direct access to the luminal (apical) surface. Apical access is necessary to allow physiologic comparison of pathogen interaction with the intestinal epithelial barrier. We hypothesize that apical-out (AO) enteroids will provide a relevant NEC model to study this relationship.MethodsFollowing the institutional review board approval (#11610-11611), neonatal intestinal tissue was collected from surgical specimens. Stem cells were collected; enteroids were generated and grown to maturity in BO conformation then everted to AO. Enteroids were untreated or treated for 24 h with 100 μg/mL lipopolysaccharide and hypoxia. Protein and gene expression were analyzed for inflammatory markers, tight junction (TJ) proteins and permeability characteristic of NEC.ResultsApical TJ protein zonula occludens-1 and basolateral protein β-catenin immunofluorescence confirmed AO configuration. Treated AO enteroids had significantly increased messenger RNA (P= 0.001) and protein levels (P< 0.0001) of tumor necrosis factor-α compared to controls. Corrected total cell fluorescence of toll-like receptor 4 was significantly increased in treated AO enteroids compared to control (P= 0.002). Occludin was found to have significantly decreased messenger RNA in treated AO enteroids (P= 0.003). Expression of other TJ proteins claudins-1, -4 and zonula occludens-1 was significantly decreased in treated AO enteroids (P< 0.05).ConclusionsAO enteroids present an innovative model for NEC with increased inflammation and gut barrier restructuring. This model allows for a biologically relevant investigation of the interaction between the pathogen and the intestinal epithelial barrier in NEC.