內毒素為脂多醣體(lipopolysaccharide;LPS)，是所有革蘭氏陰性菌外膜所共有的醣酯質(glycolipid)成分。LPS 誘發生物性反應主要為發炎反應。哺乳動物腸道黏膜的腸腺存在有為數甚多的黏液分泌細胞，形態如西洋高腳杯，因此稱為杯狀細胞(goblet cell)，功能為分泌黏液包覆在上皮表面，作為保護屏障，發炎介質會引發黏液排
放。本實驗目的在探討以高劑量內毒素誘發大鼠產生全身性發炎反應時，引起腸腺杯狀細胞之反應。發炎反應機制有多條路徑，因此本實驗進一步利用羥自由基清除劑dimethylthiourea(DMTU)前處理，探討自由基是否對於內毒素所引發的大鼠腸道腺體細胞黏液分泌造成影響。使用Sprague-Dawley 品系大鼠做為實驗對象，從股靜脈注射LPS(15mg/Kg)，數秒鐘完成注射，利用印地安墨汁做為血管血漿滲漏標定染劑，過了5 或30 分鐘後,將動物犧牲灌流,取下腸道組織，以親水性塑膠進行組織包埋、做切片，並以Alcian blue-PAS 染色觀察杯狀細胞黏液分泌程度，最後採用數位影像分析軟體(SimplePCI)進
&#64008;計&#63870;分析。實驗結果發現高劑&#63870;內毒素注射5 分鐘與30 分鐘，十二指腸腺杯&#63994;細胞黏液排放之細胞&#63849;百分比與對照組在統計學有顯著差&#63842;(P＜0.01)，增加為7-8 倍。而迴腸腸腺杯&#63994;細胞黏液排放之細胞&#63849;百分比遠高於對照組，約5-18 倍之多。但杯&#63994;細胞黏液佔腸腺上皮面積之百分比，&#63847;&#63809;十二指腸或迴腸均在30 分鐘實驗組下&#64009;為對照組的1/2，具有統計學上的顯著差(P<0.01)。另外DMTU 的抑制效應，在DMTU+LPS 30 分鐘的十二指腸杯&#63994;細胞黏液佔腸腺上皮面積之百分比與LPS 30 分鐘組比較，約為1.4 倍，（P＜0.01）。十二指腸腸腺杯&#63994;細胞黏液排放之細胞&#63849;百分比與LPS 30 分鐘組比較，
約&#64009;低90%（P＜0.01）。迴腸則在DMTU+LPS 5 分鐘與LPS 5 分鐘&#63864;組相比，杯&#63994;細胞黏液排放之細胞&#63849;百分比約&#64009;低1/2（P＜0.05）。由實驗結果推&#63809;靜脈注射高劑&#63870;內毒素可誘發腸腺體杯&#63994;細胞黏液排放，且30 分鐘效應大於5 分鐘。並且依據LPS 使腸腺杯&#63994;細胞黏
液佔上皮面積之百分比&#64009;低,得知因為mucus &#63978;開細胞質,跑進&#63930;腸腺管腔所致。而自由基清除劑DMTU 對腸腺杯&#63994;細胞黏液排放的抑制效應在十二指腸較顯著。

Endotoxin, a kind of lipopolysaccharide (LPS), is the glycolipid composition of cell walls of all Gram negative bacteria. The main biological reaction triggered by LPS is inflammation reaction. The
surface epithelium of intestinal mucosa contains a large number of goblet cells which function as secreting mucus to soak chime and form a protection for digestive tract. The purpose of this experiment was to study the variations of the mucus secretion of intestinal gland goblet cells and whether dimethylthiourea (DMTU), a hydroxyl radical scavenger, could exert an inhibitory effect at different time points after LPS application. Sprague-Dawley rats received an intravenous injection of LPS (15 mg/kg, over 5 seconds) through the femoral vein. India ink was used to label the leaky microvessels where the extravasated colloidal particles of dye were
trapped between the endothelium and basement membrane. 5 and 30 minutes after LPS injection, strips of intestinal tissues were dehydrated
and embedded with glycol methacrylate. Tissue sections 2 micrometers in thickness were histochemically stained with Alcian blue-PAS reagent. The number of goblet cells in intestinal glands of duodenum and ileum were counted. Mucus area and epithelial area of sampled glands were recorded
with SimplePCI and statistically analyzed. 5 or 30 minutes after LPS, the percent (%) of the number of discharging goblet cells in intestinal glands
significantly increased to 7-8 times as the saline control in duodenum. In the ileum, the number of discharging goblet cells in intestinal glands
significantly increased to 5-18 times as the saline control. 30 min after LPS, the percent (%) of goblet cell mucus in epithelial area of intestinal glands decreased to half the value of control. DMTU prior to LPS, the mucus-discharging ability of goblet cells was inhibited. In duodenum, DMTU pretreatment 30 min earlier than LPS, the percent (%) of goblet
cell mucus in epithelial area of intestinal glands increased to 1.4 times and the percent (%) of the number of discharging goblet cells in intestinal
glands significantly was reduced by 90%. In ileum, DMTU pretreatment 5 min earlier than LPS, the percent (%)of the number of discharging
goblet cells in intestinal glands decreased to one half as the control. From the experiment results, we could come to a conclusion that intravenous injection of a high dose of LPS induced an compound exocytotic release of mucus granules resulting in cavitation of goblet cell supranuclear cytoplasm and the released mucus temporarily accumulated in the lumen of glands. It is suggested that hydroxyl radicals were involved in LPS-induced mucus release from goblet cells.

目錄.........................................................................................1
【中文摘要】................................................................. . ...2
【Abstract】..................................................................... .4
【緒&#63809;】................................................................................6
一、內毒素(End oto x in ) ..........................................6
二、自由基(Free radicals) .....................................8
三、杯&#63994;細胞(Goblet cell).......................................10
四、Dimethylthiourea 的藥&#63972;作用................................13
【研究目的】............................................................14
【材&#63934;與方法】...........................................................15
一、實驗動物................................................................15
二、試藥與溶液配製....................................................15
三、動物實驗與標本處&#63972;..............................................17
四、小腸腺體杯&#63994;細胞黏液分&#63848;程&#64001;評估................21
五、統計分析..........................................................22
【實驗結果】................................................................23
一、注射LPS後，引起的小腸組織結構變化.................23
二、注射LPS之後，引起杯&#63994;細胞結構及功能..........23
三、DMTU前處&#63972;，對於內毒素所引發的大鼠腸道黏液分&#63848;的抑制效應.............................................................24
四、小腸腺體杯&#63994;細胞黏液佔腸腺上皮面積之百分比.................................................................................24
五、小腸腺杯&#63994;細胞黏液排放之細胞&#63849;的百分比................................................................................26
【討&#63809;】.......................................................................29
一、注射LPS後，引發小腸組織結構及細胞結構之變化...................................................................................29
二、LPS引起的杯&#63994;細胞黏液分&#63848;作用.................................................................................30
三、DMTU抑制杯&#63994;細胞之黏液排放............................32
【結&#63809;】....................................................................34
【&#63851;考文獻】.............................................................35
【圖表與圖解】...........................................................42

Adler KB, Hardwick DH, Craighead JE (1982) Porcine tracheal goblet cell ultrastructure: a three-dimensinal reconstruction. Exp Lung Res 3:69-80.


Agusti C, Takeyama K, Cardell LO, Ueki I, Lausier J, Lou YP, Nadel JA (1998) Goblet cell degranulation after antigen challenge in sensitized guinea pigs. Am J Respir Crit Care Med 158:1253-1258.


Antunes F, Han D, Cadenas E (2002) Relative contributions of heart mitochondria glutathione peroxidase and catalase to H2O2 detoxification in vivo conditions. Free Radi Biol Med 9:1260–1267.


Astiz M.E.,Rackow E.C.(1998) Septic shock .Lancet 351:1501-5


Auphan N.,DiDonato J.A.,Rosette C.,Helmberg A.,Karin M.(1995) Immunosuppression by glucocorticoids:inhibition of NF-κ B activity through induction of IκB synthesis.Science 270:286-290


Basbaum J.H.(1986) Regulation of airway secretory cells.Clinic.Chest Med 7:231-237


Blyth DI, Pedrick MS, Savage TJ, Hessel EM, Fattah D (1996) Lung inflammation and epithelial changes in a murine model of atopic asthma. Am J Cell Mol Biol 14:425-438.


Bone R.C.,Grodzin C.J.,Balk R.A.(1997) Sepsis:a new hypothesis for pathogenesis of the disease process.Chest 112:235-243


Borovikova LV, Ivanova S, Zhang M, Yang H, Botchkina GI, Watkins LR, Wang H, Abumrad N, Eaton JW, Tracey KJ (2000) Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405:458-462.

Brockhausen et al., “Enzymatic basis for sialyl-Tn expression in human colon cancer cells,” Glycoconjugate Journal, (1998) 15:595-603.

Crouser ED, Julian MW, Weisbrode SE, Dorinsky PM (1997) Endotoxin-induced ileal Vo2-Do2 alterations do not correlate with the severity of ileal injury. J Crit Care 12:83-91.


Crouser ED, Julian MW, Dorinsky PM (1999) Ileal Vo2-Do2 alterations induced by endotoxin correlate with severity of mitochondrial injury. Am J Respir Crit Care Med 160:1347-1353.


Crouser ED, Julian MW, Weinstein DM, Fahy RJ, Bauer JA (2000) Endotoxin-induced ileal mucosal injury and nitric oxide dysregulation are temporally dissociated. Am J Respir Crit Care Med 161:1705-1712.


Deitch E (1990) Bacterial translocation of the gut flora. J Trauma 30:S184-S189.


Enss ML, Muller H, Schmidt-Wittig U, Kownatzki R, Coenen M, Hedrich HJ (1996) Effects of perorally applied endotoxin on colonic mucins of germfree rats. Scand J Gastroenterol 31:868-874.


Forstner GG (1995) Signal transduction, packaging and secretion of mucins. Annu Rev Physiol 57:585-605.


Fox RB (1984) Prevention of granulocyte-mediated oxidant lung injury in rats by a hydroxyl radical scavenger, dimethylthiourea. J Clin Invest 74:1456-1464.


Furuya S, Naruse S, Hayakawa T (1998) Intravenous injection of guanylin induces mucus secretion from goblet cells in rat duodenal crypts. Anat Embryol 197:359-367.


Fujii Y, Magder S, Cernacek P, Goldberg P, Guo Y, and Hussain SN(2000) Endothelin receptor blockade attenuates lipopolysaccharide-induced pulmonary nitric oxide production.Am.J.Respir.Crit.Care Med 161:982-989


Gail D.B.,Lenfant C.J.M(1983) Cell of the lung:biology and implication.Am.Respir.Dis 34:366-387


Guo JJ, Wang DS, Huang HT (2003) Spontaneous remission of edema and regranulation of goblet cells in rat trachea after capsaicin-induced acute inflammation. Anat Embryol 206:301-309


Heumann D, Adachi Y, Roy DL, Ohno N, Yadomae T, Glauser MP and Calandra T (2001) Role of plasma, lipopolysaccharide-binding protein, and CD14 in response of mouse peritoneal exudate macrophages to endotoxin. Infection and Immunity 69:378–385.


Jacob RA (1995) The integrated antioxidant system.Nutr Res 15:755-766


Janis HJ, Elaine MB and John ER (1988) Thiourea and dimethylthiourea decrease human neutrophil bactericidal function in vitro. Inflammation 12:515-524


Kindt TJ, Goldsby RA, Osborne BA, Kuby J: “Leukocyte activation and migration” in: Immunology, 6th edition, pp327-350.


Kuo HP, Rohde JAL, Tokuyama K, Barnes PJ, Rogers DF (1990) Capsaicin and sensory neuropeptide stimulation of goblet cell secretion in guinea-pig trachea. J Physiol (Lond) 431:629-641.


LaMont JT (1992) Mucus: the front line of intestinal mucosal defense. Ann NY Acad Sci 664:190-201.


Laszlo F, Whittle BJ, Moncada S (1994) Time-dependent enhancement or inhibition of endotoxin-induced vascular injury in rat intestine by nitric oxide synthase inhibitors. Br J Pharmacol 111:1309-1315.


Laszlo F, Evans SM, Whittle BJ (1995) Aminoguanidine inhibits both constitutive and inducible nitric oxide synthase isoforms in rat intestinal microvasculature in vivo. Eur J Pharmacol 272:169-175.


Laszlo F, Whittle BJ (1997) Actions of isoform-selective and non-selective nitric oxide synthase inhibitors on endotoxin-induced vascular leakage in rat colon. Eur J Pharmacol 334:99-102.


Marshall J.C(2001) Inflammation,coagulopathy,and the pathogensis of multiple organ dysfunction syndrome.Crit.Care Med 29:99-106


McCuskey RS, Urbaschek R and Urbaschek B (1996) The microcirculation during endotoxemia. Cardiovascular Res 32:752-763.


Newman TM, Robichaud A, Rogers DF (1996) Microanatomy of secretory granule release from guinea pig tracheal goblet cells. Am J Respir Cell Mol Biol 15:529-539.


Pattanaik U and Prasad K (2001) Reactive oxygen species and endotoxic shock: effect of dimethylthiourea. Journal of Cardiovascular Pharmacology and Therapeutics 6:273-285.


Phillips TE (1992) Both crypt and villus intestinal goblet cells secrete mucin in response to cholinergic stimulation. J Physiol 262 (Gastrointest Liver Physiol 25):G327-G331.


Plaisancie P, Barcelo A, Moro F, Claustre J, Chayvialle JA, Cuber JC (1998) Effects of neurotransmitters, gut hormones, and inflammatory mediators on mucus discharge in rat colon. Am J Physiol 275 (Gastrointest Liver Physiol 38): G1073-G1084.


Read MA, Cordle SR, Veach RA, Carlisle CD and Hawiger J (1993) Cell-free pool of CD14 mediates activation of transcription factor NF-κB by lipopolysaccharide in human endothelial cells. Proc Natl Acad Sci 90:9887-9891.


Rangel-Frausto M.,Pittet D.,Costigan M.,Hwang T.,Davis C.,Wenzel R(1995) The natural history of the systemic inflammatory response syndrome(SIRS).JAMA 273:117-123


Rietschel E, Holst O, Brade L, Muller-Loennies S, Mamat U, Zahringer U, and Beckmann F (1996) Bacterial endotoxin: chemical constitution, biological recognition, host response, and immunological detoxification. Curr Top Microbiol Immunol 216:39-81.


Robbins SL: “Acute and chronic inflammation” in Pathologic basis of disease. 7th edition (2005) W.B. Saunders Company Press, pp47-86

Shimizu T, Hirano H, Majima Y, Sakakura Y (2000) A mechanism of antigen-induced mucus production in nasal epithelium of sensitized rats. A comparison with lipopolysaccharide-induced mucus production. Am J Respir Crit Care Med 161: 1648-1654.


Silverman MH (1999) Bacterial endotoxin in human disease. XOMA.


Singer M,Brealey D(1999) Mitochondrial dysfunction in sepsis.Biochem.Soc.Symp 66:149-166


Smirnova MG, Guo L, Birchall JP, Pearson JP (2003) LPS up-regulates mucin and cytokine mRNA expression and stimulates mucin and cytokine secretion in goblet cells. Cell Immunol 221: 42-49.


Specian RD, Neutra MR (1980) Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine. J Cell Biol 85:626-640.


Specian RD, Neutra MR (1982) Regulation of intestinal goblet cell secretion. I. Role of parasympathetic stimulation. Am J Physiol 242:G370-379.


Specian RD, Oliver MG (1991) Functional biology of intestinal goblet cells. Am J Physiol 260 (Cell Physiol 29):C183-C193.


Stevens A, Lowe J : “Alimentary tract” in: Histology, 1st edition, pp143-175


Tasaka S, Ishizaka A, Yamada W, Shimizu M, Koh H, Hasegawa N, Adachi Y and Yamaguchi K (2003) Effect of CD14 blockade on endotoxin-induced acute lung injury in mice. Am J Respir Cell Mol Biol 29:252-258.


Takeyama K.,Tamaoki J.,Nakata J.,Konno K(1996) Effect of oxitropium bromide on histamine-induced airway goblet cell secretion.Am.J.Respir.Crit.,Care Med.154:231-236.


Tjardes T.,Neugebaure E(2002) Sepsis research in the next millennium: concentrate on the software rather than the hardware.Shock 17:1-8


Wasil M, Halliwell B and Grootvelt M（1987）The specificity of thiourea, dimethylthiourea and dimethly sulphoxide as scavengers of hydroxyl radicals. Biochem. J 243: 867-870