Hosic Sanjin, Lake Will, Stas Eric, Koppes Ryan, Breault David T, Murthy Shashi K, Koppes Abigail N
Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, MA 02115 USA.
Division of Endocrinology, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115 USA.
Cell Mol Bioeng. 2020 Jun 24;13(5):487-505. doi: 10.1007/s12195-020-00633-0. eCollection 2020 Oct.
The intestinal epithelium contains specialized cells including enterocytes, goblet, Paneth, enteroendocrine, and stem cells. Impaired barrier integrity in Inflammatory Bowel Disease is characterized by elevated levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Prior studies in immortalized lines such as Caco-2, without native epithelial heterogeneity, demonstrate the amelioration of TNF-α compromised barrier integrity nicotinic (nAChR) or muscarinic (mAChR) acetylcholine receptor activation.
A tissue-engineered model of primary human small intestinal epithelium was derived from dissociated organoids cultured on collagen-coated Transwells. Differentiation was accomplished with serum-containing media and compared to Caco-2 and HT-29 regarding alkaline phosphatase expression, transepithelial electrical resistance (TEER), and IL-8 secretion. Inflammation was modeled basal stimulation with TNF-α (25 ng/mL) with or without nicotine (nAChR agonist) or bethanechol (mAChR agonist). Apoptosis, density (cells/cm), TEER, lucifer yellow permeability, 70 kDa dextran transport, cell morphology, and IL-8 secretion were characterized.
Primary intestinal epithelium demonstrates significant functional differences compared to immortalized cells, including increased barrier integrity, IL-8 expression, mucus production, and the presence of absorptive and secretory cells. Exposure to TNF-α impaired barrier integrity, increased apoptosis, altered morphology, and increased secretion of IL-8. Stimulation of nAChR with nicotine did not ameliorate TNF-α induced permeability nor alter 70 kDa dextran transport. However, stimulation of mAChR with bethanechol decreased transport of 70 kDa dextran but did not ameliorate TNF-α induced paracellular permeability.
A primary model of intestinal inflammation was evaluated, demonstrating nAChR or mAChR activation does not have the same protective effects compared to immortalized epithelium. Inclusion of other native stromal support cells are underway.
肠上皮包含多种特化细胞，包括肠上皮细胞、杯状细胞、潘氏细胞、肠内分泌细胞和干细胞。炎症性肠病中屏障完整性受损的特征是促炎细胞因子水平升高，包括肿瘤坏死因子-α（TNF-α）。先前在诸如Caco-2等无天然上皮异质性的永生化细胞系中的研究表明，烟碱型（nAChR）或毒蕈碱型（mAChR）乙酰胆碱受体激活可改善TNF-α损害的屏障完整性。
原发性人小肠上皮的组织工程模型源自接种于胶原包被的Transwell上的解离类器官。用含血清培养基实现分化，并就碱性磷酸酶表达、跨上皮电阻（TEER）和IL-8分泌与Caco-2和HT-29进行比较。通过用TNF-α（25 ng/mL）进行基础刺激，有或没有尼古丁（nAChR激动剂）或氨甲酰甲胆碱（mAChR激动剂）来模拟炎症。对凋亡、密度（细胞/cm）、TEER、荧光素黄通透性、70 kDa葡聚糖转运、细胞形态和IL-8分泌进行了表征。
与永生化细胞相比，原发性肠上皮表现出显著的功能差异，包括屏障完整性增加、IL-8表达增加、黏液产生以及存在吸收性和分泌性细胞。暴露于TNF-α会损害屏障完整性、增加凋亡、改变形态并增加IL-8分泌。用尼古丁刺激nAChR并不能改善TNF-α诱导的通透性，也不会改变70 kDa葡聚糖转运。然而，用氨甲酰甲胆碱刺激mAChR可降低70 kDa葡聚糖的转运，但不能改善TNF-α诱导的细胞旁通透性。
对一种原发性肠道炎症模型进行了评估，结果表明与永生化上皮相比，nAChR或mAChR激活没有相同的保护作用。目前正在纳入其他天然基质支持细胞。
