Lipopolysaccharide induced intestinal epithelial injury: a novel organoids-based model for sepsis in vitro.
Lipopolysaccharide induced intestinal epithelial injury: a novel organoids-based model for sepsis in vitro.
复制标题
脂多糖诱导的肠上皮损伤：一种基于类器官的新型脓毒症体外模型
DOI：
10.1097/cm9.0000000000002348
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发表时间：
2022-09-20
影响因子：
6.1
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中科院分区：
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类器官培养技术的进步使人们对疾病的发病机制有了更深入的了解，而这在脓毒症中以前很少研究。我们的目的是建立一个合适的基于类器官的脓毒症肠损伤模型。构建稳定传代的类器官并用脂多糖（LPS）预处理以模拟脓毒症诱导的肠损伤。使用LPS诱导的脓毒症模型作为参考。我们使用定量实时聚合酶链反应来评估炎症因子和抗菌肽的RNA水平。采用酶联免疫吸附试验（ELISA）检测蛋白质水平，苏木精-伊红染色检测小肠组织病理学变化，免疫组化和免疫荧光检测肠上皮屏障功能。Perkin Elmer Operetta™用于获得三维类器官的高分辨率图像。24小时后LPS浓度>150 μg/mL被鉴定为引起类器官生长限制。当LPS浓度>100 μg/mL时，24 h后闭合小带1和闭合素的荧光强度明显降低。LPS刺激8 h后，白细胞介素（IL）-1α、肿瘤坏死因子α、粒细胞-巨噬细胞集落刺激因子、IL-6和再生胰岛衍生蛋白3 α、β和γ的RNA表达水平升高。这些结果类似于小鼠败血症模型中肠上皮层改变的结果。对于IL-10，只有当LPS水平>200 μg/mL时24 h，RNA表达水平才增加。本研究提供了初步的肠道体外模型，以研究LPS诱导的类似脓毒症的肠道损伤的影响。该模型为免疫相关机制的探索和有效药物的筛选提供了平台。
Advances in organoid culture technology have provided a greater understanding of disease pathogenesis, which has been rarely studied in sepsis before. We aim to establish a suitable organoids-based intestinal injury model for sepsis. Stable passaged organoids were constructed and pre-treated with lipopolysaccharide (LPS) to mimic sepsis-induced intestinal injury. The LPS-induced sepsis model was used as a reference. We used quantitative real-time polymerase chain reaction to evaluate the RNA levels of inflammatory factors and antimicrobial peptides. Enzyme-linked immunosorbent assay was used to evaluate the protein levels, hematoxylin and eosin staining was used to evaluate the pathology of the small intestine of mice, and immunohistochemistry and immunofluorescence were used to evaluate the intestinal epithelial barrier function. Perkin Elmer Operetta™ was used to obtain high-resolution images of three-dimensional organoids. An LPS concentration >150 μg/mL after 24 h was identified to cause organoid growth restriction. The fluorescence intensity of zonula occludens-1 and occludins at LPS concentrations >100 μg/mL decreased significantly after 24 h. After LPS stimulation for 8 h, the RNA expression levels of interleukin (IL)-1α, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, IL-6, and regenerating islet-derived protein 3 alpha, beta, and gamma increased. These results resembled those of intestinal epithelial layer alterations in a mouse sepsis model. For IL-10, the RNA expression level increased only when the LPS level >200 μg/mL for 24 h. This study provides the primary intestinal in vitro model to study the effects of LPS-induced intestinal injury resembling sepsis. This model provides a platform for immune associated mechanism exploration and effective drug screening.