Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
Department of Endoscopy and Medicine, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.
J Crohns Colitis. 2017 May 1;11(5):621-630. doi: 10.1093/ecco-jcc/jjw186.
Patients with ulcerative colitis [UC] are at an increased risk of developing colitis-associated cancer [CAC], suggesting that continuous inflammation in the colon promotes the transformation of colonic epithelial cells. However, the mechanisms underlying cell transformation in UC remain unknown. We therefore aimed to investigate the effect of long-term inflammation on intestinal epithelial cells [IECs] using organoid culture.
IECs were isolated from mouse colon, and were cultured according to a method for a three-dimensional [3D] organoid culture. To mimic chronic inflammation, a mixture of cytokines and bacterial components were added to the medium for over a year. Cell signal intensity was assessed by 3D immunofluorescence. Cell transformation was assessed by microarray with gene set enrichment analysis.
Stimulation with cytokines resulted in a significant induction of target genes for the nuclear factor [NF]-κB pathway in colonic organoids. Following 60 weeks of continuous stimulation, cell differentiation was suppressed. Continuous stimulation also resulted in significant amplification of NF-κB signalling. Amplified NF-κB signalling by long-term stimulation remained in colonic organoids even 11 weeks after the removal of all cytokines. Some genes were specifically upregulated only in colonic organoids after the removal all cytokines following long-term stimulation.
Colonic organoids stimulated with cytokines for a prolonged period were established as in vitro model to assess long-term epithelial responses to inflammatory cytokines. Chronic inflammation led to sustained NF-κB signalling activation in colonic organoids, resulting in cell transformation that might be related to the carcinogenesis of CAC in UC.
溃疡性结肠炎[UC]患者发生结肠炎相关性癌症[CAC]的风险增加，这表明结肠内的持续炎症促进了结肠上皮细胞的转化。然而，UC 中细胞转化的机制尚不清楚。因此，我们旨在使用类器官培养来研究长期炎症对肠上皮细胞[IEC]的影响。
从小鼠结肠中分离出 IEC，并根据三维[3D]类器官培养方法进行培养。为了模拟慢性炎症，将细胞因子和细菌成分的混合物添加到培养基中持续一年以上。通过 3D 免疫荧光评估细胞信号强度。通过微阵列进行细胞转化评估，并进行基因集富集分析。
细胞因子刺激导致结肠类器官中核因子[NF]-κB 途径的靶基因显著诱导。经过 60 周的持续刺激，细胞分化受到抑制。持续刺激还导致 NF-κB 信号的显著放大。即使在去除所有细胞因子 11 周后，长期刺激引起的 NF-κB 信号放大仍保留在结肠类器官中。一些基因仅在长期刺激后去除所有细胞因子的结肠类器官中特异性上调。
用细胞因子刺激延长时间的结肠类器官被建立为体外模型，以评估长期上皮对炎症细胞因子的反应。慢性炎症导致结肠类器官中 NF-κB 信号的持续激活，导致细胞转化，这可能与 UC 中 CAC 的癌变有关。
