Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids
Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids
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DOI：
10.1016/j.scr.2019.101383
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发表时间：
2019-03-01
影响因子：
1.2
通讯作者：
Tripal, Philipp
中科院分区：
文献类型：
--
作者：
Bardenbacher, Marco;Ruder, Barbara;Tripal, Philipp
上皮屏障完整性的异常调节涉及许多消化道疾病，包括炎症性肠病和结直肠癌。肠上皮细胞类器官培养为肠屏障的体外分析提供了新的视角。然而，从二维培养物的屏障功能分析的既定方法必须调整到三维类器官结构的分析。在这里，我们描述了分析肠类器官上皮屏障功能和分子调控的方法。将具有和不具有干扰素-γ-受体2基因的上皮细胞特异性缺失的肠类器官对干扰素-γ的屏障反应用作模型系统。所建立的方法允许监测干扰素-γ诱导的活性类器官渗透性变化的动力学。蛋白水解降解和改变的紧密连接蛋白claudin-2，-7，和-15的本地化检测使用共聚焦旋转光盘显微镜与3D重建。Hessian分析用于定量密封蛋白的再定位。总之，我们提供了一种新的方法学方法，定量分析肠上皮屏障功能的三维类器官模型。
The aberrant regulation of the epithelial barrier integrity is involved in many diseases of the digestive tract, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial cell organoid cultures provide new perspectives for analyses of the intestinal barrier in vitro. However, established methods of barrier function analyses from two dimensional cultures have to be adjusted to the analysis of three dimensional organoid structures. Here we describe the methodology for analysis of epithelial barrier function and molecular regulation in intestinal organoids. Barrier responses to interferon-y of intestinal organoids with and without epithelial cellspecific deletion of the interferon-y-receptor 2 gene were used as a model system. The established method allowed monitoring of the kinetics of interferon-y-induced permeability changes in living organoids. Proteolytic degradation and altered localization of the tight junction proteins claudin-2, -7, and - 15 was detected using confocal spinning disc microscopy with 3D reconstruction. Hessian analysis was used for quantification of relocalization of claudins. In summary, we provide a novel methodologic approach for quantitative analyses of intestinal epithelial barrier functions in the 3D organoid model.