2016-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659418摘要：腸道是人類身體中共生菌最多的器官；在正常生理情況下，腸道上皮屏障會將這些共 生菌侷限於腸腔中。若上皮屏障失常，腸道菌極有可能入侵黏膜到血液循環，進而導致人體 全身性發炎和敗血併發症。在許多臨床疾病中，包括發炎性腸疾和腸阻塞，可觀察到過量的 促發炎性細胞激素以及腸道細菌轉移之病理現象。目前腸胃醫學界對腸道細菌轉移的分子機 制仍不清楚。本實驗室研究發現腸道細菌可透過兩種方式穿越上皮屏障，包括通過細胞間受 損的緊密連結空隙或經由錯入細胞的途徑(Am J Pathol 2014; Am J Physiol Gastrointestinal Hepatol 2014; Gut Pathogens 2013; Crit Care Med 2011)。過去文獻指出間細胞和跨細胞途徑皆依 賴長型肌絲輕鍵激酶long myosin light chain kinase (long MLCK)之活化；然而，其特異調 控方式尚待釐清。最近我們研究發現上皮細胞次結構中，肌動肌凝蛋白環和終末網處之肌絲輕鏈(MLC) 磷酸化皆是由long MLCK活化所造成的。已知人類上皮細胞具有兩種long MLCK剪接變異 體，差別在於第一型多出69個氨基酸序列並涵蓋Src kinase作用點；而MLC亦有三種異構 型的表現。本計畫中，我們將採用上皮細胞株和類器官培養方式觀察促發炎性細胞激素刺激 後的通透性變化，以及利用發炎性腸疾和腸阻塞之動物模式探討這些異構型如何分別調控間 細胞和跨細胞途徑，導致腸道屏障失能。本計晝研究目的是（1)利用小鼠疾病模式和人類上皮細胞株觀察促發炎性細胞激素在 上皮細胞誘發間細胞和跨細胞途徑的劑量效應和時間點，以及屏障失衡與肌絲輕鏈磷酸化之 相關性；(2)藉由基因改造小鼠模式探討MLCK/MLC異構型在終末網或圍連結肌動肌凝蛋白 環的表現量和次細胞位置的改變；（3)確認促發炎性細胞激素透過Src kinase的特異性活化和 MLCK剪接變異體之表現量調控，參與間細胞和跨細胞途徑的機制；（4)利用初代腸道類器 官和細胞株培養方式探討上皮細胞中參與細菌内吞和運送過程之胞器，及偵測促發炎性細胞 激素是否藉由一氧化氮依賴性dynamin蛋白的硝酸化引起嵌合芽接與通透增加現象。<br> Abstract: Intestinal tract is the largest reservoir of commensals in human body, of which enteric bacteria are normally lumen-confined by epithelial barrier. Once the barrier is breached, enteric bacteria may invade the mucosa and reach circulatory bloodstream, predisposing the host to systemic inflammation and septic complications. Clinical disorders, such as inflammatory bowel disease (IBD) and intestinal obstruction (IO) display high levels of proinflammatory cytokines associated with abnormal bacterial translocation. The molecular mechanisms of bacterial translocation across epithelial barrier remains incompletely understood. Studies from our laboratory demonstrated two routes of bacterial entry, including passage between impaired tight junctions or penetration through epithelial cells (Am J Pathol 2014; Am J Physiol Gastrointestinal Hepatol 2014; Gut Pathogens 2013; Crit Care Med 2011). Previous reports have indicated that both transcellular and paracellular permeability are dependent on activation of long myosin light chain kinase (long MLCK). Nevertheless, the MLCK-mediated differential regulation on paracellular and transcellular barrier changes remains elusive.Recent data indicated that phosphorylation of MLC in subcellular structures of epithelial cells, i.e. perijunctional actinomyosin ring and terminal web, are both mediated by activation of long MLCK. Two splicing variants of long MLCK with a difference of 69 amino acids corresponding to a Src kinase domain in MLCK-1 but not MLCK-2, and three isoforms of MLCs were identified in human epithelial cells. In this project, we plan to use human epithelial cell lines and organoid cultures, as well as animal models of IBD and IO, to investigate how proinflammatory cytokines differentially regulate splicing variants of long MLCK to target distinct MLC isoforms to control paracellular and transcellular permeability.The objectives of the current project are:⑴ To evaluate the dose- and time-dependent effects of proinflammatory cytokines on transcellular and paracellular bacterial influx in correlation to myosin light chain phosphorylation in mouse models and human intestinal epithelial cells; (2) To investigate the expression levels and subcellular localization of MLCK/MLC isoforms for phosphorylation at terminal web or perijunctional regions using genetically modified mice; (3) To correlate the roles of proinflammatory cytokines in differential activation of Src kinase and regulation of specific MLCK splicing variants for control of transcellular and paracellular barrier damage; (4) To characterize the transcellular endocytic pathways and investigate the involvement of cytokine-dependent nitric oxide-induced dynamin nitrosylation in the mechanisms of endocytosed vesicle budding and trafficking using cell lines and primary organoid cultures.腸道上皮屏障共生菌緊密連結細菌内吞肌絲輕鏈磷酸化激酶異構型類器官培養Intestinal epithelial barriercommensal bacteriatight junctionsbacterial endocytosismyosin light chainkinaseisoformorganoid culture