臺灣博碩士論文加值系統

腸道機械性阻塞是一種導致腹部病痛一個很重要的因素。目前已有研究指出，腸道阻塞會導致細菌轉移現象 (bacterial translocation, BT)的發生，表示腸道上皮細胞屏障已遭受破壞。已知腸道上皮細胞之肌凝蛋白輕鏈激酶 (myosin light chain kinase, MLCK)將肌凝蛋白輕鏈 (myosin light chain, MLC)磷酸化, 使圍連結肌動肌凝蛋白環 (perijunctional actinomyosin ring, PAMR)收縮, 導致細胞間通透性增加。本研究主要目的在探討MLCK是否有參與大鼠腸道機械性阻塞引起腸道黏膜組織受損及上皮細胞屏障喪失的機制。

方法與材料：本研究所使用的實驗動物為Wistar品系的雄性大鼠。分別在給予迴腸阻塞手術之前的24、12、1小時以及之後的12小時投予MLCK之抑制劑 ML-7 (1 mg/kg, i.p.)或是其載液，並且和假手術處理的大鼠做比較。迴腸阻塞手術是將大鼠的迴腸 (盲腸之前的10 cm處)綁一死結作為迴腸阻塞的實驗模組；假手術處理的組別，則是接受腹腔手術但是不給予迴腸阻塞的大鼠。手術之後的24小時，分別取出五段腸段 (十二指腸、空腸、迴腸、近端大腸以及遠端大腸)進行實驗分析。實驗的分析包括組織型態觀察、腺窩深度相對絨毛長度比值、細胞凋亡偵測、腸道骨髓過氧化酶活性測量、嗜中性白血球染色、Ussing Chamber Systems測定組織對於大分子蛋白質山葵過氧化酶 (horseradish peroxidase, HRP)的通透性，以及肝臟和脾臟組織中菌落數的量化。另外利用西方轉漬的技術偵測黏膜組織中MLC磷酸化的程度。

結果：結果顯示，迴腸阻塞會導致迴腸絨毛變寬變短以及增加腺窩深度相對於絨毛長度的比值，而絨毛頂端以及剝落在管腔中的細胞有細胞凋亡的現象。然而，迴腸阻塞和假手術處理組的大鼠之腸道骨髓過氧化酶活性以及嗜中性白血球染色並無顯著差異。此外，迴腸阻塞會導致迴腸以及近端大腸對於管腔面HRP之通透性增加，在肝臟以及脾藏也觀察到有細菌生長，證實BT的現象。迴腸阻塞後會導致迴腸黏膜中MLC的磷酸化增加，預先投與ML-7，則可以降低MLC的磷酸化及部分改善由迴腸阻塞所導致之黏膜組織傷害，並且有效抑制迴腸對於HRP
過度的通透性，然而對近端大腸通透性以及BT卻沒有影響。

總結：本篇研究探討腸道阻塞導致上皮細胞屏障破壞的分子機制，由結果可知，迴腸機械性阻塞會導致腸黏膜傷害包括絨毛變短、腺窩增生、細胞凋亡以及屏障功能的破壞，包括上皮細胞通透性增加以及腸內細菌轉移體內的現象。抑制MLCK的活化，則可以改善組織型態並且降低迴腸對大分子的通透性，表示腸阻塞引起的大分子通透性是經由上皮細胞間的途徑。而細菌轉移體內的機制則與
MLCK之活化無關。

Mechanical intestinal obstruction is one of the most common causes of abdominal emergencies. Enteric bacterial translocation (BT) has been reported in experimental obstruction models, suggesting disruption of epithelial barrier. Myosin light chain kinase (MLCK) is involved in the contraction of perijunctional actinomyosin ring (PAMR) in enterocytes and increases the transepithelial permeability via a paracellular route. The aim of this study was to investigate the role of MLCK in the mechanism of mucosal injuries and epithelial barrier defects induced by intestinal obstruction.

METHODS: Male Wistar rats (200-300g) received MLCK inhibitor ML-7 (1 mg/kg, i.p.) or vehicle at 24, 12 and 1 hr before and 12hrs after ileal obstruction. Sham-operated controls received ML-7 or vehicle but without obstruction. Obstruction animals underwent ligation of the ileum 10 cm proximal to the cecal junction. After 24 hrs post-obstruction, the duodenum, jejunum, ileum, proximal colon and distal colon were collected. Histological slides were used for the evaluation of mucosal structures and measurement of crypt/villi ratio. The level of cell apoptosis was detected by TUNEL staining, and the level of mucosal inflammation was determined by myeloperoxidase (MPO) activity and neutrophils staining. Transepithelial permeability was measured by mucosal-to-serosal horseradish peroxidase (HRP) flux on Ussing chambers. The presence of bacteria in the spleen and liver was examined using fresh blood agar culturing for BT. The phosphorylation level of MLC in protein extracts from ileal mucosal scrapings was detected by western blotting techniques.

RESULT: Intestinal obstruction induced villi shortening and widening associated with increased crypt/villi ratio in the ileum. Disrupted villi tip and increased apoptotic epithelial cells were seen in the ileal lumen after obstruction. However, the MPO level and neutrophil staining in intestinal segments of obstructive rats were not different from those in sham groups. Ileal obstruction increased the HRP flux in the ileum and proximal colon, whereas no changes were seen in other segments. The bacterial colony forming unit (CFU) in the liver and spleen in obstruction animals were significantly higher than those without obstruction, suggesting enteric bacterial translocation. In addition, obstruction resulted in the phosphorylation of MLC in ileal mucosa, that is inhibitable by ML-7 pretreatment. Pretreatment with ML-7 partially protected the tissues from obstruction-induced mucosal injuries, and inhibited the increase of HRP flux in the ileum, but not in the proximal colon. Moreover, administration of ML-7 had no effect on the enhanced BT induced by obstruction.

CONCLUSIONS: Intestinal obstruction causes villous shortening, crypt proliferation, increased mucosal cell apoptosis, and epithelial barrier defects, i.e. enhanced epithelial permeability and bacterial translocation. Inhibition of MLCK reduced the increased HRP flux in the ileum, suggesting that obstruction-induced increase of transepithelial macromolecular permeability is mediated via a paracellular route. Bacterial translocation induced by ileal obstruction is MLCK-independent. The understanding of the molecular mechanism in epithelial barrier defects may shed light to the development of therapeutic intervention for intestinal obstructive injuries.

口試委員會審定書-------------------------------------------------------Ⅰ
致謝-------------------------------------------------------------------------Ⅱ
中英文縮寫名詞對照表-------------------------------------------------Ⅲ
中文摘要-------------------------------------------------------------------Ⅳ
英文摘要-------------------------------------------------------------------Ⅵ

壹、文獻回顧--------------------------------------------------------------1
一、 腸阻塞----------------------------------------------------------------1
1.1 腸阻塞之臨床症狀及診斷------------------------------------------1
1.2 腸阻塞之動物模式及病理現象------------------------------------3
二、 腸道屏障功能--------------------------------------------------------5
2.1 物理性屏障 (physical barrier)-------------------------------------6
2.2 化學性 (chemical barrier)及免疫性屏障 (immune
barrier)------------------------------------------------------------------------12
三、 目的---------------------------------------------------------------------13

貳、材料與方法-------------------------------------------------------------15
一、 實驗動物---------------------------------------------------------------15
二、 實驗設計---------------------------------------------------------------15
三、 迴腸阻塞手術過程---------------------------------------------------16
四、 肌凝蛋白輕鏈激酶 (MLCK)抑制劑的藥物投予---------------17
五、 細菌轉移現象 (bacterial translocation, BT)測定-------------17
六、 組織切片及染色------------------------------------------------------18
6.1 檢體的製備--------------------------------------------------------------18
6.2 蘇木紫-伊紅染色 (Haematoxylin and Eosin Staining)--------18
6.3 組織損傷評量 (histological damage scoring)-------------------19
6.4 絨毛長度及腺窩深度測量---------------------------------------------19
6.5 細胞凋亡染色------------------------------------------------------------19
6.6 嗜中性白血球 (neutrophils)染色------------------------------------20
七、 上皮組織電生理儀器 (Ussing Chamber System)-------------21
7.1 腸組織架設---------------------------------------------------------------21
7.2 腸道上皮組織電生理測定---------------------------------------------21
7.3 腸道上皮組織通透性測定---------------------------------------------22
八、 腸道組織骨髓過氧化酶(myeloperoxidase, MPO)活性測定-23
九、 磷酸化肌凝蛋白輕鏈蛋白 (pMLC)的西方轉漬法--------------24
9.1 黏膜層蛋白質萃取-------------------------------------------------------24
9.2 總蛋白質定量-------------------------------------------------------------24
9.3 電泳-------------------------------------------------------------------------24
9.4 免疫轉漬------------------------------------------------------------------25
十、 統計分析-----------------------------------------------------------------25

參、結果------------------------------------------------------------------------.27
第一部分實驗：迴腸阻塞 (IO)對於腸道的影響------------------------27
一、 腸道組織型態-----------------------------------------------------------27
二、 細胞凋亡程度-----------------------------------------------------------27
三、 腸道骨髓過氧化酶活性以及嗜中性白血球數量-----------------28
3.1 腸道骨髓過氧化酶活性-------------------------------------------------28
3.2 嗜中性白血球的偵測----------------------------------------------------28
四、 迴腸阻塞對於腸道上皮細胞屏障功能的影響--------------------28
4.1 腸道上皮組織之電生理------------------------------------------------.28
4.2 腸道上皮組織之通透性-------------------------------------------------29
五、 迴腸阻塞對於細菌轉移現象 (BT)的影響-------------------------29

第二部分實驗：肌凝蛋白輕鏈激酶 (MLCK)抑制劑對於迴腸阻塞腸
道的影響。----------------------------------------------------------------------29
一、 MLCK抑制劑對於腸道組織型態的影響---------------------------29
二、 MLCK抑制劑對細胞凋亡程度的影響------------------------------30
三、 MLCK抑制劑對於迴腸之上皮細胞屏障喪失的影響------------30
3.1 腸道上皮組織之電生理--------------------------------------------------30
3.2 腸道上皮組織之通透性--------------------------------------------------30
四、 MLCK抑制劑對於迴腸阻塞導致BT現象的影響----------------31
五、 磷酸化肌凝蛋白輕鏈 (pMLC)的表現-------------------------------31

肆、討論--------------------------------------------------------------------------32

圖表及說明-----------------------------------------------------------------------.40

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