葡聚餹硫酸鈉誘發腸炎模式中腸道上皮細胞穿細胞和間細胞通透性的改變

Transcellular and Paracellular Permeability Changes of Intestinal Epithelium in Dextran Sodium Sulfate-induced Enterocolitis

翁儷庭(Li-Ting Weng)

指導教授 ： 余佳慧

國立臺灣大學/醫學院/生理學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602220

摘要

背景:發炎性腸疾(IBD)之致病原因包含腸道屏障失常和細菌入侵。細菌可藉由穿細胞以及間細胞途徑進入腸道上皮細胞，然而其機制目前不甚了解。葡聚餹硫酸鈉 (DSS)是一種化學物質常用來誘發小鼠產生腸炎，在過去in vitro實驗中高濃度(>3%)之DSS會直接對腸道上皮細胞造成毒殺性，然而低濃度(1~3%)之DSS會造成緊密連結損壞且未有細胞凋亡。近期新發現的促炎性激素TL1A在發炎性腸疾病患的腸道組織表現量上升，而在本實驗室in vitro實驗中發現TL1A會活化腸道上皮細胞之MLCK而促使肌凝蛋白輕鏈磷酸化，導致細菌內吞增加。目的: 探討葡聚餹硫酸鈉誘發腸炎模式中穿細胞和間細胞通透性增加的時間點，以及TL1A和MLCK在穿細胞途徑中扮演的角色。 方法材料: BALB/c小鼠飲用含2.5% DSS水0、1、4和7天。測量組織骨髓過氧化酶活性、組織腸炎評分、細胞凋亡情形和細菌轉移至脾臟和肝臟的數量。利用Ussing chamber測定腸道通透性，及抗硫酸慶大黴素試驗進行腸道上皮細胞中內吞細菌數定量。以螢光原位雜交實驗觀察腸內細菌分布。部分動物組別在飲用DSS前進行腹腔施打MLCK抑制劑(ML-7)和中和性TL1A之抗體。此外，給予long MLCK基因剔除小鼠飲用DSS水進行研究。 結果: 小鼠飲用DSS 7天後，體重減輕且大腸長度縮短。在飲用DSS 4天觀察到組織發炎程度增加、腸炎評分增加和腸道上皮細胞凋亡上升，而細菌轉移至脾臟和肝臟之數量也顯著增加。DSS飲用4天後會造成腸道大分子通透性上升，然而在飲用DSS 1天後即可觀察到腸道上皮細胞會細菌內吞的現象。另外，飲用DSS後也會造成黏膜層中TL1A、磷酸化MLC和切割態occludin蛋白表現量上升，而TL1A、TNFα和IFNγ mRNA表現量也增加。利用腹腔注射中和性TL1A抗體和ML-7後會抑制大腸上皮細胞中細菌內吞，以及MLC、IκB、Akt磷酸化的現象。與野生型小鼠相比，long MLCK基因剔除小鼠在飲用DSS 4天後, 迴腸和大腸中dextran通透性、切割態occludin表現量、細胞凋亡等現象較低，且細菌轉移減少。 結論: 飲用DSS後會造成腸道上皮細胞屏障缺失包含MLCK依賴性的穿細胞和間細胞通透性增加，而後伴隨發炎反應。此外，TL1A有參與MLCK活化引起腸道上皮細胞的細菌內吞作用。

關鍵字

葡聚?硫酸鈉誘發腸炎模式 ； 發炎性腸疾 ； 腸道通透性 ； 腫瘤壞死因子激素1A ； 肌凝蛋白輕鏈激?

並列摘要

Background: Impaired gut barrier function and enteric bacterial influx are involved in the pathogenesis of inflammatory bowel diseases (IBD). Mechanisms of abnormal bacterial influx via transcellular and paracellular pathways across epithelium remain incompletely understood. Dextran sodium sulfate (DSS) is a widely used chemical to induce colitis in mice, of which in vitro studies showed that high dose (>3%) causes epithelial cytotoxicity whereas low dose (1~3%) induces cell death-independent tight junctional disruption. A novel proinflammatory cytokine, TNF-like lA (TL1A), was found to increase in intestinal tissues of IBD patients. Our pilot study in vitro demonstrated that TL1A induced bacterial endocytosis and myosin light chain phosphorylation via a myosin light chain kinase (MLCK)-dependent pathway in epithelial cells. Aim: To investigate the timing of transcellular and paracellular barrier damage, as well as to explore the roles of TL1A and MLCK in regulation of transcellular permeability in DSS-induced enterocolitis model. Methods: BALB/c mice were given 2.5% DSS in drinking water for 0, 1, 4 and 7 days. Intestinal myeloperoxidase (MPO) activity, histopathological score, TUNEL-positive cells, bacterial translocation into spleen and liver tissues were determined. Intestinal permeability was measured on Ussing chamber. Intracellular bacterial counts in isolated epithelial cells were determined using a gentamycin resistance assay. Bacterial presence in mucosal tissues was determined by fluorescent in situ hybridization (FISH). In some groups, mice were intraperitoneally injected with ML-7 (a MLCK inhibitor) or neutralizing anti-TL1A prior to DSS water drinking. Moreover, long MLCK-KO mice were also given DSS water for experiments. Results: Body weight loss and shortened colon length were observed after drinking DSS for 7 days. Increased MPO activity, histopathological damage, epithelial apoptosis, and bacterial translocation were observed after drinking DSS for 4-7 days. Elevated luminal-to-serosal macromolecular dextran flux in gut tissues was found after DSS drinking for 4 days, whereas bacterial endocytosis by epithelial cells was noted after 1 day. Mucosal samples showed increased protein levels of TL1A, phosphorylated MLC, and cleaved occludin, and mRNA levels of TL1A, TNFα and IFNγ after drinking DSS. Increased bacterial endocytosis by colonic epithelial cells and MLC phosphorylation after drinking DSS for 1 day were inhibited by ML-7 or neutralizing anti-TL1A. Compared to wild type mice, long MLCK-KO mice showed less dextran flux, occludin cleavage, and epithelial death in the colon, associated with decreased bacterial translocation after drinking DSS for 4 days. Conclusion: Epithelial barrier dysfunction, including increase in MLCK-dependent transcellular and paracellular permeability, preceded gut mucosal inflammation in DSS-induced enterocolitis model. Moreover, TL1A is also involved in MLCK-dependent bacterial endocytosis by intestinal epithelial cells.

並列關鍵字

DSS-induced enterocolitis ； IBD ； Intestinal permeability ； TL1A ； MLCK

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