【中文摘要】

內毒素為一種脂多醣 (lipopolysaccharide, 簡稱為LPS)，是來自革蘭氏陰性菌細胞壁的毒性化學物質，可刺激免疫細胞 (包含巨嗜細胞，白血球) 釋放細胞介素 (cytokine)，這些物質包括interleukin-1β、interleukin-6 及tumor necrosis factor-α等，這些發炎前媒介物進而引起全身性急性發炎反應，以及個體多重器官衰竭等敗血性休克症狀。而在發炎反應的過程中，血管系統的血漿滲漏是一個重要的指標。其他已被廣泛研究的發炎物質如：辣椒素、物質P及組織胺，可刺激整個呼吸道及部分消化道引起發炎反應，是由於它們可以誘發微血管後小靜脈內皮形成許多直徑約1.4 μm的內皮隙，從此處漏出大量血漿，蓄積在結締組織而形成水腫。哺乳類的消化系統的上皮組織存在大量的杯狀細胞，其分泌的黏液除了浸潤食糜之外，亦可形成一道屏障，保護消化道的上皮組織免於受到物理及化學性的傷害。在發炎反應、病原菌的感染或是其他的免疫因子的刺激下，均會改變杯狀細胞的功能及其數目。本實驗的重點在於：(1) 研究靜脈注射高劑量LPS (15 mg/kg) 對於小腸血漿滲漏程度及杯狀細胞分泌作用的影響。(2) 迷走神經及膽鹼性接受器是否參與LPS所引起的血漿滲漏及杯狀細胞細胞的分泌作用。本實驗利用靜脈注射發炎追蹤劑印地安墨汁來標定血漿滲漏的血管，並且利用銀染色灌流法來標定內皮細胞間的內皮隙；將小腸組織以 3μm 厚度的切片及Alcian blue-PAS 染色法來呈現黏液中的醣蛋白，用以觀察杯狀細胞的分泌作用。實驗結果顯示，靜脈注射LPS不僅引起小腸組織的血漿滲漏並且伴隨著高比率的杯狀細胞分泌作用，造成微血管後小靜脈及收集靜脈形成大量的內皮隙。而血漿滲漏在LPS刺激的早期 (5到30分鐘) 即大量產生，但是杯狀細胞的分泌則出現在內毒素刺激後30分鐘，並且在1小時後黏液會大量的堆積在絨毛間隙中。以muscarinic 接受器的拮抗劑atropine前處理，可明顯的抑制LPS所引起杯狀細胞的分泌。然而，以atropine處理或是施以雙側頸迷走神經切除術，對抑制LPS所引起的血漿滲漏並不一致。由實驗結果推論，靜脈注射高劑量的內毒素所引起的敗血性休克中，小腸的血漿滲漏及杯狀細胞的過度分泌是有時間性的，並且伴隨著膽鹼性muscarinic接受器的活化。

【Abstract】

Lipopolysaccharide (LPS) is the toxic chemical component of the cell wall in all gram-negative bacteria which can stimulate immune cells, including macrophages and white blood cell, to release cytokines such as interleukin-1β, interleukin-6 and tumor necrosis factor-α. These pro-inflammatory mediators induce systemic acute inflammation and multiple organs dysfuction syndrome in sepsis. Plasma leakage from microvasculature is a hallmark of inflammation. Previous studies have demonstrated that other inflammatory agents, such as capsaicin, substance P and histamine could cause the acute formation of numerous endothelial gaps in the venules that result in extensive plasma leakage in the inflammatory tissues of the whole respiratory tract and a part of digestive tract in a few minutes. Mammalian intestines have many goblet cells that synthesize mucus and discharge it into the intestinal lumen. The mucus film that covers the surface epithelium facing the lumen of digestive system, is an immune defense that can prevent gastrointestinal epithelium from chemical and physical damage and act as a lubricant. Changes in goblet cell function and number are involved in microbial infection, inflammatory syndromes and immune factors. This study was aimed to investigate: (1) The degree of Plasma leakage and goblet cell hypersecretion in the small intestine of rats after an intravenous injection of a high dose of LPS (15 mg/kg), and (2) The involvement of vagus nerve and cholinergic receptors in plasma leakage and goblet cell secretion. For the study of plasma extravasation in small intestine during endotoxema, India ink was used as the tracer to mark the inflamed leaky microvessels. Rats were perfusion-fixed through the aorta, and endothelial gaps between endothelial cells of blood vessels were made visible with silver staining. The methacrylate sections of the ileum 3 μm in thickness were stained with Alcian blue and periodic acid-schiff reagent to detect glycoproteins of goblet cells. Our results showed that LPS not only caused an increase in plasma leakage but also triggered degranulation of many goblet cells in villi and crypts. Numerous gaps were found in postcapillary venules and collecting venules, and plasma extravasation was observed in the serosa and tunica muscularis rat small intestine after LPS. Extensive plasma extravasation occurred in earier phases (5-30 min). However, numerous goblet cells started to discharge mucus granules 30 min after LPS treatment. A large amount of extracellular mucus was accumulated between intestinal villi 1 hour after LPS stimulation. Pretreatment with atropine, the muscarinic receptor antagonist, significantly inhibited goblet cell secretion. The inhibitory effect of pretreatment with atropine or bilateral cervical vagotomy on LPS-induced plasma leakage was not consistent. It is concluded that the plasma leakage and goblet cell hypersecretion induced by endotoxin shock was time-dependent and was associated with activation of muscarinic cholinergic receptors.

目錄 (Index)

【中文摘要】---------------------------------------------------------------1
【Abstract】---------------------------------------------------------------3
【緒論】-------------------------------------------------------------------5
【研究目的】---------------------------------------------------------------13
【材料與方法】-------------------------------------------------------------14
【實驗結果】---------------------------------------------------------------21
【討論】-------------------------------------------------------------------26
【結論】-------------------------------------------------------------------31
【參考文獻】---------------------------------------------------------------32
【圖】---------------------------------------------------------------------41

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