臨床與疾病動物模式中腸道屏障功能探討

Abstract

研究背景：氣囊輔助小腸鏡，利用可漲縮的氣囊在小腸內膨脹固定並套疊腸道，來達成深入插入小腸檢查的目的，是臨床上新穎的內視鏡檢查工具。吾人已知腸道內有著多樣的腸內菌。膨脹的氣囊對於腸道的刺激是否會引發局部腸道血流改變與腸道屏蔽功能的變化，進而引發腸內細菌轉移至周邊血液菌血症之風險，目前未有相關研究。此外，腸道屏蔽功能在大腸腫瘤新生過程中，究竟扮演何種角色，目前仍未有定論。 目的：本研究旨在（1）前瞻觀察性臨床研究，觀察氣囊輔助小腸鏡術檢查患者群中，是否有腸內細菌轉移菌血症之細菌培養或細菌基因標記之證據；創新設計氣囊小腸鏡之動物實驗模式，以小鼠大腸內氣囊膨脹刺激來探究氣囊膨脹造成腸道組織缺氧與腸道屏蔽功能之影響；以細胞株探究短期與長期缺氧對於上皮細胞通透性之影響，（2）研究大腸腫瘤新生與腸道上皮屏蔽功能的相關性。 研究方法及結果：第一部分：共有十三位患者納入臨床觀察研究，經簽署知情同意書後，氣囊小腸鏡術前與術後均採周邊血液做細菌內毒素定量與細菌核酸檢測。十三位中有四位患者在術後測得血液中發現細菌核酸。術後的周邊血液內毒素含量比術前周邊血液內毒素含量為高。然而，未發現有臨床上發燒或敗血症症狀。為模擬氣囊小腸鏡，小鼠經肛門插入含氣囊之小兒科矽膠導尿管，對大腸腸段做氣囊膨脹刺激。我們觀察到當氣囊膨脹時，大腸組織發生局部組織缺氧變化，當氣囊收縮時，大腸組織回覆供氧狀態。當腸段受到長時間的氣囊膨脹刺激，會使得腸道的通透性有增加之傾向。在近端大腸受到短時間的氣囊膨脹，會產生顯著的腸道屏蔽增強之現象。此外，人類大腸上皮細胞株Caco-2細胞缺氧5-20分鐘之後，呈現上皮緊密結合組成增加，使得細胞株上皮屏蔽增強；另一方面，長期缺氧則會破壞細胞株上皮通透性。第二部分：腸道通透性已知受到腸道上皮細胞之肌動蛋白輕鏈蛋白激酶而調控。在致癌劑與化學性腸炎藥劑誘發大腸腫瘤新生之小鼠動物模型中，探究原生種小鼠與肌動蛋白輕鏈蛋白激酶基因缺失小鼠之腸道通透性與大腸腫瘤新生個數與腫瘤面積：發現在致癌劑肌動蛋白輕鏈蛋白激酶基因缺失小鼠中，在腫瘤發生前已有觀察到腸道通透性下降。而在大腸腫瘤新生數目與大腸腫瘤總面積，基因缺失小鼠遠較於原生種小鼠為高。 結論：氣囊小腸鏡術後患者中偶可見無症狀之內毒素血症與菌血症，經氣囊短暫膨脹造成之腸道缺氧可能引發保護性的腸道屏蔽增強，然而長期的腸道缺氧則會破壞腸道屏蔽功能。臨床醫師於操作氣囊小腸鏡時，應避免長時間氣囊膨脹刺激腸道而破壞腸道屏蔽功能，以避免細菌轉移感染之風險。另一方面，腸道上皮細胞之肌動蛋白輕鏈蛋白激酶參與了腸道屏蔽與腸道腫瘤新生的調控，可能扮演腫瘤抑制基因的新穎功能。本研究證實腸道屏蔽在調節腸道恆定功能上扮演著舉足輕重的影響。

Background: Human intestines harbor abundant microbes, which rely on a monolayered epithelial barrier to prevent bacterial translocation or gut-derived inflammatory and septic complications. The procedure of balloon-assisted enteroscopy (BAE) utilizing distensible balloons inside the small intestines to facilitate deep endoscopy. It remains elusive whether balloon distension causes perturbation of blood flow and elicits risk of bacterial translocation. Moreover, the relationship between gut epithelial hyper-permeability and colon cancer development has yet to be explored. The aims of thesis researches: (1) To gather clinical microbiological and molecular evidence of bacterial translocation by BAE in patients and to establish a murine model of colonic balloon distension to investigate tissue hypoxia and intestinal barrier, and (2) To assess the relationship between epithelial barrier changes and colon tumor formation. Methods and Results: Part I: Thirteen patients were enrolled for BAE procedures after informed consents, and blood samples were obtained before and after BAE for paired comparison. Four of the 13 patients (30.8%) had positive bacterial DNA in blood after BAE. Post-BAE endotoxemia was higher than pre-BAE level. No clinical symptom of sepsis or fever was reported. To mimic clinical BAE, mice were subjected to colonic balloon distension. Tissue oxygen levels were measured by relative concentration of oxygenated and deoxygenated hemoglobin. Local tissue hypoxia was observed during balloon inflation, and reoxygenation after deflation. A trend of increased gut permeability was seen after long-term distension, whereas a significant reduction of permeability was observed by short-term distention in the proximal colon. Part II: Intestinal permeability is regulated by activation of epithelial myosin light chain kinase). Decreased luminal-to-serosal macromolecular flux and bacterial endocytosis in epithelial cells were observed in colonic tissues of MLCK-deficient mice compared to wild type mice at the early phase of mutagen induction. At the late phase, the tumor numbers and areas in MLCK-deficient mice were higher than those in wild types. Conclusion: Sporadic cases of bacteremia were found after BAE, without septic symptoms. Short-term hypoxia by balloon distension yielded a protective effect whereas long-term hypoxia caused damage on gut barrier. Moreover, epithelial MLCK is involved in regulation of intestinal permeability and tumorigenesis. Epithelial barrier function plays an indispensable role in maintenance of gut homeostasis.