以電刺激或辣椒素刺激分佈於呼吸道中的感覺神經，可使C-fiber感覺神神經元的神經末梢釋放物質P及CGRP等神經傳遞物質，物質P可與血管內皮細胞上的NK-1受器結合，產生神經性發炎反應，包括由血管中滲漏血漿以及水腫的形成(Lundberg and Saria, 1983；McDonald et al., 1988)。Evans blue為一水溶性染劑，由於與血漿中的蛋白質具有高度親合力，因此常被使用在血漿滲漏的追蹤染劑，將Evans blue由組織中萃取出來進行定量分析，用以評估觀察血漿蛋白質滲漏的程度。鉀離子通道活化劑可以抑制位於呼吸道與膀胱的神經性血漿滲漏(Hollywood et al., 1998)，然而以Evans blue直接刺激人類臍靜脈上皮細胞上的鈣離子活化的鉀離子通道上，對微血管通透性同樣有所影響(Wu et al.,1999)，這可能意味著動物體實驗同樣能影響血管通透性。先前研究指出Evans blue能抑制由辣椒素所誘導的咳嗽以及支氣管收縮(Bolser et al.,1995)，因此假設以Evans blue前處理可能對呼吸道神經性發炎具有生理效應。我們以物質P與辣椒素誘導呼吸道神經性發炎再以膠體追蹤劑India ink作為評估影響的程度，觀察在不同濃度的Evans blue (0, 3, 15,及30 mg/ml/kg)前處理下，對物質P與辣椒素所誘導的呼吸道血漿滲漏及水腫是否具有影響？同時也觀察位於氣管的漿液細胞超顯微結構，在Evans blue前處理下對物質P誘導的呼吸道發炎是否有所改變。實驗結果顯示：在物質P的部分，高濃度Evans blue (30 mg/ml/kg)作用下，會使物質P造成的印地安染劑標示的血管面積密度降低下來，在氣管部分減少十分之七以上(P<0.01)；在左主支氣管部分被標示的血管面積密度減少十分之七以上(P<0.01)；在右主支氣管部分被標示的血管面積密度減少十分之七左右(P<0.01)，然而水腫的抑制作用在統計學上並無差異(P>0.05)。在辣椒素誘導呼吸道神經性發炎的部分，高濃度Evans blue (30 mg/ml/kg)作用下，被印地安染劑標示的血管相較於只給予saline前處理，在氣管部分減少十分之七以上 (P<0.01)；在左主支氣管部分被標示的血管面積密度減少十分之七以上(P<0.01)；在右主支氣管部分被標示的血管面積密度減少十分之七左右(P<0.01)，而在抑制水腫的部分，高濃度Evans blue (30 mg/ml/kg)對照saline前處理發現，水腫部分降低十分之八以上，為顯著差異 (P<0.01)。在漿液細胞表面變化與統計方面，以Evans blue 30 mg/ml/kg前處理組別對照saline前處理組別，在物質P 誘導下，Evans blue 30 mg/ml/kg組別的活化漿液細胞數目明顯少於saline前處理組別，減少幅度為十分之七以上(P<0.01)。因此我們認為，高濃度Evans blue能抑制物質P與辣椒素所誘導的血漿滲漏以及辣椒素所引起的水腫，這是由於活化large-conductance Ca2+-activated K+ channels，但對物質P所引起的水腫則無明顯抑制作用，低濃度Evans blue則可能會增強呼吸道神經性發炎作用。掃描式電子顯微鏡下發現，物質P確實能引起呼吸道黏膜上皮漿液細胞活化，而在高濃度的Evans blue前處理下，活化漿液細胞數目明顯減少，確實有抑制漿液細胞活化的作用。

Stimulation of C-fiber sensory neurons innervating the respiratory tract with electricity or capsaicin leads to the liberation of substance P, CGRP and other neuropeptides from the nerve terminals. Substance P (SP) binds to the NK-receptors on the membrane of vascular endothelial cells and elicits neurogenic inflammatory responses. These inflammatory responses include plasma leakage and the subsequent edema formation (Lundberg and Saria, 1983；McDonald et al., 1988). Evans blue is a hydrophilic dye and is often used as a tracer of plasma leakage due to its’ high affinity to the plasma proteins. Plasma leakage causes Evans blue extravasates from the blood vessels and remains in the tissues. The more plasma leaks from the blood vessels, the more Evans blue will extravasate into the tissues. Measuring extravasated Evans blue dye that is extracted from tissues, is useful for evaluation of the amount of plasma leakage. Potassium channel openers can inhibit neurogenic plasma leakage in the airways and urinary bladder (Hollywood et al., 1998). Evans blue directly stimulates large-conductance Ca2+-activated K+ channels in cultured endothelial cells of human umbilical vein (Wu et al., 1999). This suggests that it may influence the permeability of the microvessels in vivo. A previous study shows that Evans blue dye blocks capsaicin-induced cough and bronchospasm in the guinea pig (Bolaer et al., 1995). We postulated that pretreatment with Evans blue may influence the extent of neurogenic inflammation in the rat airways induced by the application of either SP or capsaicin. India ink was used as a colloidal tracer dye to label the leaky vessels. The present study investigated whether different concentration of Evans blue (0, 3, 15 and 30 mg/ml/kg) pretreatment could affect the plasma leakage and edema formation in rat lower airways in response to intravenous injection of either SP or capsaicin. The amount of plasma leakage was expressed by the area density of India ink-labeled leaky blood vessels. We also investigated whether Evans blue influenced the ultra-structural change in tracheal serous cells induced by intravenous injection of SP. Our results showed that pretreatment with high concentration of Evans blue reduced more than seven tenths of the area density of plasma leakage in the trachea caused by SP application (P<0.01); reduced more than seven tenths in the left main bronchus (P<0.01) and reduced about seven tenths in the right main bronchus (P<0.01), compared to the control group that received saline prior to SP. However, no statistical significance was observed in edema ratio between any two groups (P>0.05). In the neurogenic inflammation of the airways caused by injection of capsaicin, pretreatment with high concentration of Evans blue reduced more than seven tenths of the area density of plasma leakage in the trachea (P<0.01); reduced more than seven tenths in the left main bronchus (P<0.01) and reduced about seven tenths in the right main bronchus (P<0.01), respectively, compared to the control group that received saline prior to capsaicin. Pretreatment with high concentration of Evans blue prior to capsaicin also reduced more than eight-tenth in edema ratio (P<0.01). In the ultra-structure change of serous cells and the stastical analysis of the number of active serous per 1000μm2 of tracheal epithelium, Evans blue pretreatment prior to SP significantly reduced the number of active serous cells by seven tenths (P<0.01) as compared to control group that received saline prior to SP. Therefore, we concluded that pretreatment with high concentration of Evans blue exerted its’ effect by opening large-conductance Ca2+-activated K+ channels and inhibited the plasma leakage induced by SP or capsaicin. But no significant inhibition was observed in edema formation induced by SP application. Low concentration of Evans blue might enhance the neurogenic inflammation of the airway. Under the observation with SEM, we found that SP activated serous cells in airway epithelium, and high concentration of Evans blue pretreatment lowered the secretory activity of serous cells. Therefore Evans blue might inhibit the activation of serous cells.

中文摘要---------------------------------------------------------1
Abstract----------------------------------------------------------3
前言---------------------------------------------------------------6
研究目的--------------------------------------------------------14
材料與方法-----------------------------------------------------15
實驗結果--------------------------------------------------------20
討論--------------------------------------------------------------26
結論--------------------------------------------------------------32
參考文獻--------------------------------------------------------33
圖表與圖解-----------------------------------------------------41

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