臺灣博碩士論文加值系統

睡眠呼吸中止症會伴隨著間歇性低氧，進而造成喉部發炎與喉氣道過度反應。喉氣道過度反應被認為是因發炎反應導致上喉辣椒素敏感型神經致敏化，造成呼吸道反射反應的增強。在近期的研究中顯示，神經滋養因子，尤其是nerve growth factor (NGF)以及brain-derived neurotrophic factor (BDNF)可促進喉部發炎作用，進而於喉氣道過度反應發展中扮演著重要的角色。然而NGF和BDNF是否參與在間歇性低氧所誘導大鼠的喉氣道過度反應中，其機制仍未明瞭。本實驗是將清醒的大鼠暴露於重複75秒一個循環的間歇性低氧中或是暴露於空氣之中，每天六小時，持續十四天，並且分別於有或無給予anti-NGF抗體、anti-BDNF抗體與IgG處理。於第十五天，在喉部給予化學刺激物(如：辣椒素、苯基二鳥胺酸與α,β-亞甲基三磷酸腺苷)，並觀察喉部受到刺激後引發的呼氣暫停反射反應，視為喉氣道過度反應之程度。在本篇研究結果顯示，有暴露間歇性低氧的大鼠相較於空氣控制組的大鼠，其對於化學刺激物所引起的呼氣暫停反應有明顯的延長；而這個反應能夠被上喉神經以周邊神經辣椒素處理法或是上喉神經剪斷來阻斷。並且，此擴大化學刺激物引發呼氣暫停現象，於大鼠經由anti-NGF抗體處理後，可有效地被抑制。另外，若給予anti-BDNF抗體處理的大鼠經過暴露間歇性低氧後，於苯基二鳥胺酸以及α,β-亞甲基三磷酸腺苷的刺激下引發呼氣暫停增強之反應，有被抑制的現象；但辣椒素所引起的擴大呼氣暫停反應，其減弱效果未達統計上的差異。在生物化學的分析中，大鼠暴露間歇性低氧後，與空氣控制組相比，其喉部組織的NGF與BDNF蛋白質表現量會提升；另外，於喉部組織脂質過氧化測試中顯示其氧化壓力也有所提升。間歇性低氧增加喉部脂質過氧化之作用，於前處理anti-NGF抗體可有效減緩，但anti-BDNF的效果則不佳。再者，若是前處理IgG的間歇性低氧大鼠，其各項測驗與分析則與單獨間歇性低氧組相差無異。從各項實驗結果顯示，暴露間歇性低氧十四天可能導致上喉辣椒素敏感型神經致敏化，進而造成喉部對於化學刺激物所引起的呼氣暫停反應明顯增強，並且神經滋養因子正向調控作用參與於此致敏化過程中。

Obstructive sleep apnea (OSA), manifested by intermittent hypoxia (IH), is associated with laryngeal airway hyperreactivity (LAH) and laryngeal inflammation. LAH is the augmented airway reflexes resulting from the sensitization of superior laryngeal capsaicin-sensitive afferents under laryngeal inflammation. Recent studies have demonstrated that neurotrophins, particularly nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), play a critical role in prompting laryngeal inflammation resulting in the development of LAH. However, whether NGF and BDNF involve in IH-induced LAH in rats remain unclear. Conscious rats were exposed to repetitive 75 seconds cycles of IH or room air (RA) for 6 h/day for 14 consecutive days with/without daily treatment with anti-NGF antibody, anti-BDNF antibody or IgG. At 16 h after their last exposure, the reflex apneic responses to laryngeal provocations with chemical stimulants (e.g. capsaicin, phenylbiguanide, and ,-methylene-ATP) were measured to reflect laryngeal reflex reactivity. Our results showed that laryngeal provocations of three chemical stimulants induced augmentation of reflex apneic response in the IH-exposed rats as compared with the RA rats, which was blocked by sectioning of the superior laryngeal nerves or perineural capsaicin treatment of superior laryngeal nerves. The augmented apneic responses to three stimulants were inhibited by treatment with anti-NGF antibody in IH-exposed rats. Additionally, the enhanced apneic responses to phenylbiguanide and ,-methylene-ATP, but not capsaicin, were significantly suppressed by treatment with anti-BDNF antibody. Biochemical analysis demonstrated that rats exposed to IH displayed increased laryngeal levels of protein expression of NGF and BDNF, and lipid peroxidation (an index of oxidative stress), compared with those exposed to RA. The increased laryngeal lipid peroxidation was significantly prevented by treatment with anti-NGF antibody, but not by anti-BDNF antibody. Treatment with IgG exerted no significant effect on the IH-induced responses. These results suggest that IH exposure for 14 days sensitizes superior capsaicin-sensitive laryngeal afferents, leading to an exaggerated apneic reflex to stimulants; this sensitizing effect is mediated through upregulation of neurotrophins.

目錄
壹、 背景知識 .................................................................................... 3
(一) 重要性 ................................................................................................................ 3
(二) 間歇性低氧 ...................................................................................................... 4
(三) 間歇性低氧促進發炎反應 ....................................................................... 6
(四) 間歇性低氧導致喉氣道過度反應(laryngeal airway
hyperreactivity, LAH) .................................................................................... 7
(五) 上喉感覺神經-上喉辣椒素敏感型 C 纖維 ....................................... 8
(六) 呼吸道中的神經滋養因子 ..................................................................... 10
(七) 間歇性低氧提高神經滋養因子之功能 ............................................ 12
二、 研究目的 .................................................................................. 15
貳、 實驗材料與方法 ......................................................................... 17
一、 研究方法 .................................................................................. 17
(一) 實驗動物 ......................................................................................................... 17
(二) 間歇性低氧模式 .......................................................................................... 17
(三) 動物的麻醉及插管 ..................................................................................... 18
(四) 生理參數測量 ............................................................................................... 19
(五) 喉氣道反射反應之測試 .......................................................................... 19
(六) 周邊神經辣椒素阻斷法( Perineural capsaicin treatment, PCT)
20
(七) 西方墨點法 (Western blotting) ........................................................... 21
(八) 喉組織脂質過氧化反應之測定 ........................................................... 23
二、 藥物製備 .................................................................................. 25
三、 實驗步驟 .................................................................................. 28
四、 資料分析與統計 ...................................................................... 30
參、 實驗結果 ..................................................................................... 31
肆、 討論 ............................................................................................. 35
伍、 結論 ............................................................................................. 41
陸、 圖表及說明 ................................................................................. 42

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