靜脈注射高劑量的內毒素(例如lipopolysaccharide, LPS)會造成試驗動物的內毒素血症，並進一步引起產生細胞介素、自由基、血漿滲漏、系統性發炎反應，然而內毒素造成的血漿滲漏是否和血管內皮隙的形成有關連目前並不清楚。在正常的生理狀態之下，腸道表面會形成一層保護障壁來抵抗腸道內的毒性物質，例如細菌和內毒素。腸道上皮的障壁失常是敗血症病患的一個重要臨床指標。在注射內毒素之後，迴腸黏膜會被破壞，迴腸絨毛上皮的細胞有超顯微結構的損傷，而細胞壞死也是很重要的特點，然而，黏液分泌細胞的反應則往往被忽略了。本研究主要目的有三點：(一)研究施打內毒素之後大鼠大腸血漿滲漏是否與內皮隙的形成有關；(二)研究內毒素是否會造成大鼠小腸黏膜絨毛和藉由複合胞吐作用(compound exocytosis)來增加杯狀細胞的分泌；及(三)探討氫氧自由基在內毒素誘發杯狀細胞分泌以及血漿滲漏中扮演之角色。
首先藉由銀染色法來展現大鼠大腸的微循環，並使用印地安墨汁作為微循環血漿滲漏程度的標示劑，觀察內毒素誘發發炎反應之後是否有內皮隙在血管內皮細胞之間形成，並探討內毒素造成的血漿滲漏是否和內皮隙形成有正相關。實驗結果顯示在注射生理食鹽水的對照組大鼠中，盲腸和結腸每1000μm2微血管後小靜脈和收集小靜脈內皮面積的內皮隙密度約為0.2 ± 0.1 ~ 0.4 ± 0.1個/1000 μm2 (n = 5)。注射內毒素5分鐘後的內皮隙密度遽然增加為12.1 ± 1.6 ~ 27.5 ± 2.2個/1000μm2 (n = 5 or 6)，為對照組的43 ~ 69倍之多(P < 0.01)。與此同時，血漿滲漏結果顯示注射內毒素5分鐘後在盲腸和結腸滲漏血管面積密度為對照組的7.8 ~ 8.2倍(P < 0.01)。血漿滲漏的異常增加和大量的內皮隙出現持續了至少30分鐘，然後到60分鐘後則降為對照組的數值(P > 0.05)。此結果說明內毒素引起的腸道血漿滲漏和微循環小靜脈內皮隙的形成有密切關係。
其次，為了正確得知黏膜絨毛上皮杯狀細胞的確實數目，本實驗發展了一套創新、有效的實驗方法，先在解剖顯微鏡下將腸道切割成約兩排絨毛的小片段，然後以掃瞄式電子顯微鏡來觀察，並以黏液物質組織化學染色來進一步確認杯狀細胞。利用數位影像分析軟體SimplePCI來測量計算小腸絨毛的上皮表面積以及杯狀細胞的數目，杯狀細胞排放程度以形成空腔的杯狀細胞數目來表示。另外利用氫氧自由基的清除劑-二甲基硫尿素(dimethylthiourea)前處理來探討氫氧自由基在內毒素誘發杯狀細胞分泌以及血漿滲漏的機轉中所扮演之角色。在掃描式電子顯微鏡的幫助之下，可以觀察到大鼠在注射內毒素之後，杯狀細胞經由複合胞吐作用排放出黏液素而形成具有空腔的杯狀細胞。在注射內毒素的5分鐘及30分鐘之後，每mm2迴腸絨毛上皮分別有693 ± 196個(n = 6)和547 ± 213個(n = 6)空腔化杯狀細胞，分別是對照組數目的5.1和8.4倍(P < 0.05)。在注射內毒素5分鐘後的十二指腸和迴腸，以及注射內毒素30分鐘後的迴腸，絨毛上杯狀細胞產生空腔化的數目 百分比均明顯上升(P < 0.05)。但如果以氫氧自由基清除劑–二甲基硫尿素前處理，形成空腔化的杯狀細胞數目與對照組相比則無統計學差異(P > 0.05)。由以上實驗結果可以得到一個結論，在內毒素誘發大鼠小腸發炎反應的急性期，氫氧自由基有參與內毒素誘導杯狀細胞複合胞吐作用活性上升以及血漿滲漏增加的機轉。

Intravenous application of a high dose of endotoxin, such as lipopolysaccharide (LPS), results in endotoxemia and sepsis in experimental animals. LPS induces production of cytokines and free radicals, plasma leakage and systemic inflammation. But the relationship between LPS-induced plasma leakage and endothelial gap formation is still unknown. Under normal physiological and pathological conditions, the mucus of intestine plays an important role in host defense mechanism as a barrier to prevent invasion of bacteria and endotoxin. The integrity of the intestinal epithelium is an important determinant of clinical outcome in septic patients. It is reported that, after LPS application, ileal mucosa is injured consequently. Necrosis of epithelial cells is also prominent feature in the villus epithelium. However, the response of mucin-secreting goblet cells is often ignored. The present study was designed to prove (1) whether LPS application increased plasma leakage by endothelial gap formation in rat intestinal tract, (2) whether LPS application increased goblet cell secretion by compound exocytotic activity in mucosal villi of small intestine; and (3) whether hydroxyl radicals were involved in LPS-induced compound exocytosis in goblet cells and plasma leakage.
First, the microcirculation of large intestine in rats was shown by using silver nitrate staining method, and India ink was used to label the leaky microvessels to express the magnitude of plasma leakage. Endothelial gaps formed between endothelial cells in the venules after LPS-induced inflammation were investigated by light and scanning electron microscopy. In saline control, the number of endothelial gaps per 1000 μm2 endothelium of postcapillary and collecting venules was 0.2 ± 0.1 ~ 0.4 ± 0.1 / 1000 μm2 (n = 5). At 5 minutes after LPS application, the endothelial gap density drastically increased to 12.1 ± 1.6 ~ 27.5 ± 2.2 / 1000 μm2 (n = 5 or 6), about 43-69 times (P < 0.01) as much as control. At the same time, the magnitude of plasma leakage, expressed by area density of India ink-labeled blood vessels, in the cecum and colon of LPS-treated rats increased to 7.8-8.2 times (P < 0.01) as much as control. Unusually high degree of plasma leakage and high number of endothelial gaps persisted for at least 30 minutes after treatment. Then, a significant reduction to the baseline level occurred at 60 minutes after LPS application (P > 0.05). The results evidently indicated that LPS-induced intestinal plasma leakage and the endothelial gap formation of venules were closely related.
In the following experiment, in order to obtain an actual number of goblet cells in the mucosal epithelium, an innovative and effective experimental method was developed and adopted to prepare small intestine specimens in this study. Tissue pieces with two rows of mucosal villi were taken under a dissecting microscope. Then, scanning electron microscope was used to observe goblet cells and histochemistry staining was applied to further identify mucosubstance. The degree of goblet cell secretion in the villus epithelium of the duodenum and ileum was expressed by the number of cavitated goblet cells undergoing compound exocytosis. Digital morphometric software SimplePCI was employed to measure the epithelial surface area of sampled villi and to count the number of goblet cells. In addition, hydroxyl radical scavenger – dimethylthiourea (DMTU) was also applied to explore the role of hydroxyl radicals involving in LPS-induced goblet cells secretion and plasma leakage. From scanning electron microscopy study, the numbers of cavitated goblet cells per mm2 of ileal villus epithelium in rats at 5 and 30 minutes after LPS injection were 693 ± 196 (n = 6) and 547 ± 213 (n = 6), respectively, which were 5.1 and 8.4 times (P < 0.05) compared with the number of saline control. The percentage of villus cavitated goblet cell numbers, in both duodenum and ileum 5 minutes after LPS and in the ileum 30 minutes after LPS, increased significantly (P < 0.05). When DMTU was given prior to LPS, the number of cavitated goblet cells and the amount of plasma leakage was inhibited and remained at the level as control (P > 0.05). It is concluded that the mechanism of the LPS-induced increase in compound exocytotic activity of goblet cells and increase in plasma leakage during acute phases of inflammatory response in rat small intestine was associated with hydroxyl radicals.

致謝-------------------------------------------------------------------i
中文摘要------------------------------------------------------------iii
Abstract-------------------------------------------------------------vi
目錄-----------------------------------------------------------------viii
圖表目錄------------------------------------------------------------ x
Abbreviations--------------------------------------------------- xiii
第一章 文獻回顧及研究目的------------------------------------1
一、發炎反應------------------------------------------------------1
二、內毒素---------------------------------------------------------3
三、血漿滲漏與內皮隙------------------------------------------9
四、杯狀細胞與黏液素分泌-----------------------------------12
五、二甲基硫尿素的抑制效果--------------------------------19
六、研究目的-----------------------------------------------------21
第二章 材料與方法----------------------------------------------23
一、化學試劑及藥物的製備-----------------------------------23
二、實驗動物-----------------------------------------------------29
三、內毒素誘發大腸血漿滲漏程度變化以及內皮隙數目變化之相關研究--------------------------------------------------30
四、內毒素誘發小腸血漿滲漏程度變化以及絨毛杯狀細胞黏液素分泌程度變化之相關研究--------------------------37
五、二甲基硫尿素對於內毒素誘發的小腸血漿滲漏及杯狀細胞黏液素分泌之抑制作用相關研究--------------------43
六、資料的統計分析--------------------------------------------44
第三章 實驗結果-------------------------------------------------45
一、內毒素誘發大腸血漿滲漏程度變化以及內皮隙數目變化之相關研究結果--------------------------------------------45
二、內毒素誘發小腸血漿滲漏程度變化以及絨毛杯狀細胞黏液素分泌程度變化之相關研究結果以及二甲基硫尿素對於內毒素誘發的小腸血漿滲漏及杯狀細胞黏液素分泌之抑制作用相關研究結果----------------------------------52
第四章 討論-------------------------------------------------------58
一、內毒素誘發內皮隙形成以及血漿滲漏之機轉--------58
二、內毒素造成杯狀細胞黏液素分泌之機轉以及二甲基硫尿素的抑制作用-----------------------------------------------67
三、結論-----------------------------------------------------------75
第五章 參考文獻-------------------------------------------------76
圖與表--------------------------------------------------------------91
相關研究成果---------------------------------------------------159
附錄---------------------------------------------------------------162

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