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標題: | 腸道寄生蟲感染小鼠之痛覺和屏障功能變化 Nociceptive and Barrier changes in Mouse intestines after Parasitic Infection |
作者: | Hsiu-Wei Wu 吳秀薇 |
指導教授: | 余佳慧(Chia-Hui Yu) |
關鍵字: | 腸躁症,精神壓力,梨形蟲,內臟高敏感性,腸道屏障, irritable bowel syndrome,psychological stress,Giardia lamblia,visceral hypersensitivity,gut barrier, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 背景與目的:
腸躁症是一種腸胃道功能性失調疾病,特徵是長期反覆性腹痛伴隨排便習慣改變,卻沒有結構上的損傷和病原體的發現。目前腸胃道醫學界對腸躁症的病理機制尚不清楚,大部分的研究是在病人身上,而無公認之動物模式。腸躁症的症狀可能會在生活壓力事件和病原感染導致腸胃炎之後出現。病原感染後出現腸躁症症狀,特稱為後感染性腸躁症,最近在挪威的研究中顯示40-80% 受到一種寄生蟲─梨形蟲感染後的病人會發展成腸躁症。先前動物實驗顯示精神壓力會增加腹痛和腸道屏障功能降低。本研究目的是利用梨形蟲感染後結合避水壓力以雙重因子刺激以建立腸躁症的小鼠模式,評估其腹痛和對腸道屏障功能的影響,並研究其致病機制。 材料與方法: 小鼠給予灌食梨形蟲 (Giardia lamblia) 的滋養體並且計數在小腸中滋養體的數目來確認感染情形。未感染組給予PBS灌食。梨形蟲定殖在小腸中數目的高峰期在感染後第4-7天,定義為「感染期」;而在兩星期後排除體外,因此定義第21-49為「後排除期」。接著進一步在後排除期中第35天進行為期十天的避水壓力 (water avoidance stress, WAS) 或無處理(non-handled, NH)。評估「感染期」與「後排除期」的腸道屏障功能與腹痛敏感程度,並探討相關分子在腸道與背根神經節中的表現。 結果: G. lamblia「感染期」之高峰在第4-7天,小腸定殖之滋養體數目最高且有屏障失能及總細菌量上升的現象;大腸則沒有影響。在「後排除期」中利用結直腸撐張刺激觀察腹部動器反應,發現感染後排除之單一因子會引起痛覺閾值降低,也就是有痛覺異常(allodynia)的現象;而單一精神壓力因子下,則有痛覺過度(hyperalgesia)的現象。在雙重因子下會產生協同效應,使兩種現象都發生。在腸 III 道屏障功能方面,腸道大分子物質通透性和黏膜層緊密連結occludin的片斷化在小腸中的G. lamblia 組別(WAS和NH)相較於個別感染組皆有增加,且在雙重因子下會使occludin片斷化更加明顯;在大腸則都沒有變化。利用螢光原位雜合技術和抗生素阻抗試驗來定位和量化腸道內吞細菌,發現小腸和大腸在G. lamblia 組別皆有細菌入侵腸組織的現象,且雙重因子對細菌入侵的影響更大。利用西方轉漬法測定腸組織與背根神經節中與腸道神經敏感化相關分子:CCK, CCK-1R (AR), CCK-2R (BR) 以及mast cell tryptase的表現。發現大腸組織之CCK在單一精神壓力、G. lamblia 後排除以及雙重因子的組別,表現量皆有明顯上升;而小腸之CCK-1R表現量在單一因子與雙重因子下表現量上升,且G. lamblia 後排除可以強化精神壓力的表現。小腸和大腸組織之CCK-2R則各組之間都沒有變化。此外,分析小腸和大腸的mast cell tryptase表現量發現G. lamblia 組別都有表現量上升,且在大腸中G. lamblia 後排除會強化精神壓力的影響。最後, CCK、CCK-1R、 CCK-2R在背根神經節中的表現量在各組之間則都沒有變化。 結論: 本實驗 G.lamblia 後排除附加慢性避水壓力的動物模式中有腹痛敏感化、腸道屏障失能以及腸道共生菌叢入侵組織等類似腸躁症的病理現象。而腸組織中CCK、CCK-1R 以及mast cell tryptase 表現量的改變可能參與其中的機制。 Backgrounds and Aims: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by chronic abdominal pain and altered bowel habits without macroscopic abnormality and presence of pathogen. The pathogenesis of IBS is still unclear and most studies are based on patient samples while animal models are not widely recognized. Symptoms of IBS may begin following stressful events or infectious gastroenteritis termed post-infectious (PI) IBS. A recent study in Norway showed that 40-80 % of patients developed IBS symptoms after a parasite Giardia infection. Previous animal studies have shown that psychological stress increases abdominal pain and intestinal barrier disruption. The aim is to establish a IBS model using post-giardiasis combined with water avoidance stress (WAS) to evaluate abdominal pain and gut barrier function. Materials and Methods: Mice were inoculated with Giardia lamblia trophozoites, and the trophozoites in the small intestine were enumerated. The number of colonized Giardia peaked on day 4-7 which was termed “colonization phase”; the parasites were cleared by day 14, and therefore, day 21-49 was denoted “post-clearance phase”. Further water avoidance stress (WAS) or nonhandled (NH) protocols were performed post-giardiasis on PI day 35. Gut barrier function and visceral hypersensitivity are experimented in “colonization V phase” and “post-clearance phase”. Besides, pain-related molecules expression in intestines and dorsal root ganglia (DRG) are analyzed. Results: Intestinal colonization of Giardia trophozoites peaked on day 4-7 and increased macromolecular permeability and tight junction occludin cleavage was seen along with commensal bacterial overgrowth. During the post-clearance phase, increased visceral motor response (VMR) was observed after low-volume colorectal distension, suggesting that post-infection alone triggers allodynia. On the other hand, increased VMR by high-volume distension was noted after WAS, indicating stress alone caused hyperalgesia. Dual factors caused synergistic effects of hyperalgesia and allodynia. Moreover, small intestinal tight junction disruption and increased epithelial permeability were seen in post-giardiasis with or without stress and augmented by dual factors of post-infection and stress. Fluorescence in situ hybridization and gentamycin resistance assay revealed the presence of bacteria in lamina propria and increased endocytosed bacteria in mucosal cells in mice post-giardiasis, which was potentiated by stress. Furthermore, the expression of pain-related molecules including CCK, CCK-1R (AR), CCK-2R (BR) and mast cell tryptase in intestine and dorsal root ganglia were analyzed by Western blotting. In both single and dual factors treated mice, we found increased CCK expression in large intestine and increased CCK-1R VI expression in small intestine, while post-giardiasis augmented the stress effect. Intestinal CCK-2R levels did not change among group. The levels of mast cell tryptase increased in both small and large intestine in post-giardiasis plus stress. The CCK, CCK-1R and CCK-2R levels in dorsal root ganglia were comparable among groups. Conclusion: Post-infection combined with stress caused visceral hypersensitivity, intestinal barrier dysfunction and commensal bacteria invasion. Intestinal CCK, CCK-1R and mast cell tryptase may be involved in the mechanisms of the IBS-like pathology. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60982 |
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