江伯倫Chiang, Bor-Luen臺灣大學:口腔生物科學研究所王友正Wang, Yu-ChengYu-ChengWang2010-05-042018-07-092010-05-042018-07-092009U0001-2907200923391300http://ntur.lib.ntu.edu.tw//handle/246246/178623氣喘是現在工業化社會常見的慢性呼吸道發炎性疾病，在許多開發中國家或已開發國家中非常普遍，主要造成氣喘的原因是遺傳因子、環境影響以及生活型態。氣喘的發生主要是T輔助型細胞的不正常偏向Th2細胞型免疫反應所導致，其症狀為間歇性與可逆性之呼吸道阻礙造成之週期性氣喘、胸腔緊繃與呼吸急促、支氣管過度敏感以及慢性的呼吸道發炎等。有許多研究指出類鴉片接受器位於免疫細胞上，參與調節免疫反應。納美芬與納美芬酮是一種類鴉片受體的對抗劑，可以透過阻斷類鴉片受體來達到抗發炎效果。所以本實驗主要目的是探討納美芬與納美芬酮在氣喘動物模式中抑制小鼠呼吸道發炎反應的效果。體外實驗中發現，經LPS刺激後的樹突細胞與巨噬細胞以納美芬與納美芬酮處理，可以使得IL-6、IL-12、IL-10與TNF-α的表現量明顯降低，顯示出納美芬與納美芬酮可以有效的抑制發炎相關的細胞激素表現。所以進一步，想利用卵清蛋白誘導小鼠的氣喘動物模式去研究納美芬與納美芬酮在動物實驗中之抗發炎效果，將實驗小鼠分為七組，每組約5–6隻，各組分別以不同方式處理。小鼠犧牲後，分析其呼吸道阻力、肺功能、引起發炎相關的細胞激素及趨化因子與卵清蛋白專一性抗體可以發現，在動物實驗中納美芬與納美芬酮可以有效抑制呼吸道阻力產生，且可以減少在肺沖洗液中與發炎相關的細胞激素與趨化因子eotaxin、IL-6與TNF-α之分泌，以及抑制嗜酸性白血球之聚集現象，但是卻不會影響血清中抗原專一性的抗體分泌與肺沖洗液中的IL-5分泌，而在脾臟細胞之IL-5、IL-10與IL-12之分泌與細胞增生反應的狀況亦不會受到影響。因此可推論納美芬與納美芬酮應可以透過阻斷類鴉片接受器方式抑制發炎反應，改善氣喘中呼吸道的發炎反應，但是卻不會影響免疫反應的走向。但是由於詳細的作用機制仍然不明確，所以未來仍有待進一步的研究去確認。Asthma is one of the most common airway inflammatory disease in the industrialized society and is the prevalence is very high in developed and developing countries. It might be caused by many factors including genetic factors, environmental factors and life style. The immune mechanism of asthma is that T helper cells are abnormally differentiated to Th2 immune response. The symptoms of asthma are intermittent and reversible airway obstruction leading to recurrent episodes of wheezing, breathlessness, chest tightness, and cough; bronchial hyperresponsiveness (BHR), which is defined as an increased sensitivity to bronchoconstrictors such as histamine or cholinergic agonists; and airway inflammation. Many researches have indicated that opioid receptors are found on immune cells, and participate in regulation of immune response. Naltrexone and nalmefene are antagonists of opioid receptors, and they can suppress inflammation with blocking the opioid receptors. For this reason, the aim of this study is to find out if naltrexone and nalmefene could suppress airway inflammation in OVA-induced murine model of asthma. The secretion of IL-6, IL-12, IL-10 and TNF-α of dentritic cells and macrophages stimulated with LPS were decreased by treatment with naltrexone and nalmefene. These results showed that naltrexone and nalmefene can reduce the secretion of cytokines related with inflammation. Further, in the OVA-induced murine model of asthma, analyzing the AHR, lung fuction, cytokines and chemokines which is related to inflammation suggesting that naltrexone and nalmefene can suppress the AHR and eosinophils assembling, and reduce the production of cytokines and chemokines connected with inflammation such as eotaxin, IL-6 and TNF-α in BALF. However, naltrexone and nalmefene can not affect the levels of antigen-specific antibodies in serum and the production of IL-5 in BALF, and have no effect in the levels of IL-5, IL-10 and IL-12 produced by splenocytes. Therefore, the results of study suggest that naltrexone and nalmefene can suppress the inflammation by blocking the opioid receptor, and reduce the airway inflammation in asthma, but have no effect to change the immune response. Finally, the mechanisms are not clear, so it needs futher investigation further in the future.口試委員審定書………………………………………………………………………1謝……………………………………………………………………………2文摘要………………………………………………………………………4文摘要……………………………………………………………………… 5寫對照表…………………………………………………………………………7錄…………………………………………………………………………… 8一章 研究背景…………………………………………………………… 11-1. 氣喘疾病之介紹……………………………………………………… 12-2. 衛生假說………………………………………………………13-3. 氣喘疾病的致病過程………………………………………………… 13-4. 氣喘疾病的產生機制……………………………………………… 14-5. 氣喘疾病的調控………………………………………………………… 15-6. 參與氣喘疾病的細胞與IgE抗體及其扮演之角色…………………… 18-7. 氣喘動物模式的建立………………………………………………… 20-8. 治療氣喘的方式…………………………………………………… 20-9. 類鴉片接受器的介紹………………………………………………… 22-10. Naltrexone與Nalmefene之介紹………………………………… 23二章 研究動機與目的…………………………………………………… 25三章 探討納美芬與納美芬酮在樹突細胞及巨噬細胞培養中的抗發炎效果………………………………………………………………… 27-1. 研究方法與材料………………………………………………… 28-1-1. 小鼠來源……………………………………………………… 28-1-2. 培養樹突細胞………………………………………………… 28-1-3. 培養巨噬細胞………………………………………………… 29-1-4. 螢光流式細胞儀…………………………………………………29-1-5. 測量細胞激素…………………………………………………30-1-6. 統計方法………………………………………………………… 31-2. 實驗結果……………………………………………………………… 31-2-1. 樹突細胞的培養狀況……………………………………………31-2-2. 巨噬細胞之培養狀況…………………………………………32-2-3. Naltrexone與Nalmefene對以LPS刺激的樹突細胞之細胞激素分泌量之影響…………………………………………………………… 32-2-4. Naltrexone與Nalmefene對以LPS刺激的巨噬細胞之細胞激素分泌量之影響…………………………………………………………… 33四章 探討納美芬與納美芬酮在氣喘之小鼠動物模式中的抗發炎效果………………………………………………………………………34-1. 研究方法與材料……………………………………………………… 35-1-1. 小鼠來源………………………………………………………… 35-1-2. 建立對OVA過敏之氣喘動物模式及治療……………………… 35-1-3. 測量對OVA有專一性之血清抗體……………………………… 36-1-4. 呼吸道阻力測試……………………………………………………37-1-5. 肺沖洗液之收集………………………………………………… 38-1-6. 肺沖洗液中血球分類計算……………………………………… 38-1-7. 肺沖洗液中細胞激素及發炎介質分泌量之測量…………………38-1-8. 脾臟細胞之收集取得………………………………………………40-1-9. OVA專一性之脾臟增生反應………………………………………40-1-10. OVA專一性抗原刺激脾臟細胞之細胞激素測量…………………41-1-11. 統計方法……………………………………………………………42-2. 實驗結果…………………………………………………………………42-2-1. 氣喘動物模式的OVA專一性抗體IgE、IgG1及IgG2a的表現狀況……………………………………………………………………42-2-2. Naltrexone與Nalmefene對於呼吸道阻力的治療效果………43-2-3. 以Naltrexone與Nalmefene治療過後小鼠肺部之細胞激素與發炎介質的分泌量變化…………………………………………………43-2-4. 以Naltrexone與Nalmefene治療過後對小鼠肺部之嗜酸性白血球聚集的影響……………………………………………………44-2-5. 以Naltrexone與Nalmefene治療小鼠過後對OVA專一性脾臟細胞增生反應之影響………………………………………………44-2-6. 以Naltrexone與Nalmefene治療小鼠過後對OVA專一性刺激之脾臟細胞的細胞激素分泌量影響………………………………44五章 討論………………………………………………………………………46…………………………………………………………………………………52考文獻……………………………………………………………………………72application/pdf2165348 bytesapplication/pdfen-US氣喘類鴉片接受器抗發炎反應納美芬納美芬酮asthmaanti-inflammationopioid receptornaltrexonenalmefenehttp://ntur.lib.ntu.edu.tw/bitstream/246246/178623/1/ntu-98-R95450014-1.pdf