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系統識別號 U0026-0812200910425418
論文名稱(中文) N-Acetylcysteine抑制家塵蟎激發小鼠之肺臟發炎反應
論文名稱(英文) N-Acetylcysteine Suppresses Dermatophagoides farine-induced Pulmonary Inflammation In Mice
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 91
學期 2
出版年 92
研究生(中文) 陳美鳳
研究生(英文) Mei-Feng Chen
學號 s4690108
學位類別 碩士
語文別 中文
論文頁數 65頁
口試委員 指導教授-余俊強
口試委員-蔡肇基
口試委員-黎煥耀
中文關鍵字 氧化性壓迫  塵蟎  氣喘 
英文關鍵字 Reactive oxygen species  asthma  Dermatophagoides farinae 
學科別分類
中文摘要 過敏性氣喘是一種慢性呼吸道疾病,目前有許多研究指出,局部微環境中的活性氧化物(Reactive oxygen species, ROS)能造成細胞的氧化性傷害,極有可能參與致敏的過程,而且在肺臟發炎過程中,體內氧化劑與抗氧化劑的不平衡,是引發細胞損害的一重要因素。N-Acetylcysteine (NAC)屬於intracellular thiol antioxidants,可以透過增加細胞內glutathione的濃度、降低NF-kB活性或透過直接清除free radical而達到抗氧化的能力。因此我希望利用兩種家塵蟎致敏小鼠的氣喘動物模式:分別是以週邊致敏模式和經呼吸道致敏模式來探討是否可藉NAC去除氧化性傷害,而抑制家塵螨所引發的肺部過敏性發炎反應。
首先在周邊致敏模式中,Der f/Alum致敏小鼠後肺泡沖洗液細胞總數、肺泡巨噬細胞、淋巴球、嗜中性球和嗜酸性球都明顯增加,且產生大量的O2-,顯示小鼠肺部處於慢性發炎反應,小鼠血清中Der f特異性IgG1抗體也明顯增加,表示免疫反應偏向Th2型。致敏小鼠經塵蟎激發後給予NAC會減少肺泡沖洗液中細胞浸潤情形,血液中的嗜酸性球亦減少,但是血清中Der f特異性IgG1抗體以及淋巴球的增生都沒有減少,顯示NAC能抑制Der f所引發的發炎反應,但是無法調控塵蟎的致敏過程。
在呼吸道致敏模式中,Der f同樣也會引起小鼠肺部發炎反應、血液中產生大量O2-和血清中Der f特異性IgG1抗體增加,若持續給予NAC則能抑制小鼠肺部的細胞浸潤情形、減低血液中O2-的產生和肺沖洗液中Nitrite、IL-6、TNF-α的產生。推測NAC可能是藉由改變血液和肺泡環境中活性氧化物濃度,以及IL-6、TNF-α的釋放,來減低小鼠肺部發炎。
英文摘要 Reactive oxygen species (ROS) play a prominent role in the pathogenesis of various inflammatory airway disorders, including asthma. N-acetylcysteine (NAC), a thiol antioxidants and free radical scavenger, can increase the intracellular glutathione concentration and decrease the NF-kB activity. The objective of my study is to clarify the role of ROS in allergic response in house dust mite Dermatophagoides farinae (Der f)-induced asthma models.
The effects of NAC have been tested in two Der f-induced asthma models: a parental sensitization model with Der f in the prescence of Alum, and an airway sensitization model with repetitive Der f intratracheal challenge. The Der f-induced responses were examined and compared between NAC-treated and non-treated mice, including inflammatory mediator production (ROS, cytokine and NO ), BAL cell infiltration, blood eosinophil count, serum antibody concentration, lung lipid peroxidation and airway hyperresponsiveness.
In the parental sensitization model, NAC could decrease total cell numbers, macrophages infiltration and reduce eosinophil counts in blood. In the single Der f challenge model, NAC could decrease total cell numbers, macrophages and lymphocyte infiltration. It also slightly inhibited nitrite, TNF-α and IL-6 contents in BAL fluid and reduced blood superoxide anion production. In the airway sensitization model, NAC suppressed the influx of total cell numbers, macrophages and TNF-α and IL-6 contents in BAL fluid. My results indicated that NAC could suppress lung inflammation of Der f-sensitized mice, probably via the reduction of blood superoxide anion production and inhibition of cytokine release.
論文目次 中文摘要……………………………………………………………………….. I
英文摘要……………………………………………………………………….. II
目錄…………………………………………………………………………….. III
圖表目錄……………………………………………………………………….. V
第一章 緒論………………………………………………………………….. 1
第一節 氣喘病之流行病學……………………………………………….. 1
第二節 家塵蟎過敏原及其致敏性……………………………………….. 1
第三節 過敏性氣喘致病機轉…………………………………………….. 3
第四節 氧化性傷害與氣喘的關係……………………………………….. 4
第五節 研究動機………………………………………………………….. 5
第二章 實驗設計…………………………………………………………….. 7
第一節 整體實驗構想…………………………………………………….. 7
第二節 具體實驗目標…………………………………………………….. 7
第三節 週邊致敏模式的動物實驗設計………………………………….. 7
第四節 呼吸道致敏模式的動物實驗設計……………………………….. 10
第三章 材料與方法………………………………………………………….. 12
第一節 方法……………………………………………………………….. 12
A、萃取家塵蟎抗原(Dermatophagoids farinae)…………………….. 12
B、實驗動物……………………………………………………………… 13
C、家塵蟎乳化抗原致敏小鼠…………………………………………… 13
D、氣管內接種(Intratracheal inoculation)……………………………. 13
E、支氣管肺泡沖洗液(Bronchoalveolar lavage,BAL)…………….. 14
F、支氣管肺泡沖洗液中白血球分類計算……………………………… 14
G、血中嗜酸性球的計數………………………………………………… 15
H、測定血清中家塵蟎特異性IgG1、IgG2a/2b抗體含量………………... 15
I、測定肺泡沖洗液或巨噬細胞刺激後上清液中細胞激素的含量……... 16
J、測定血清或肺泡沖洗液中NO的含量……………………...……….… 16
K、測定全血過氧化物(O2-)的含量 …………………………………….. 17
L、測定肺臟中lipid peroxidation的含量………………………………… 17
M、N-acetylcysteine (NAC)的使用…………………………………….… 18
N、小鼠支氣管過度反應指標……………..………………………………. 18
O、頸部淋巴結細胞以PHA及塵蟎刺激實驗…………………………….. 18
P、統計方法……………………………………………………………….. 19
第二節 材料………………………………………………………………… 19
A、 儀器設備……………………………………………………………….. 19
B、 藥品……………………………………………………………………... 20
第四章 實驗結果……………………………………………………………….. 25
第五章 討論…………………………………………………………………….. 34
參考文獻………………………………………………………………………….. 37
圖表……………………………………………………………………………….. 40

Figure 1. 已致敏小鼠在Der f激發後腹腔給予低劑量NAC的肺部發炎反應與血液中噬酸性球數目…………………………………………….. 40
Figure2. 已致敏小鼠在Der f激發後腹腔給予低劑量NAC的淋巴結細胞增生指標………………………………………………………………. 41
Figure3. 已致敏小鼠在Der f激發後腹腔給予低劑量NAC的肺泡沖洗液中TNF-α濃度………………………………………………………….. 42
Figure4. 已致敏小鼠在Der f激發後腹腔給予低劑量NAC的血清中Der f特異性IgG1和IgG2a/2b抗體的產生…………………………………. 43
Figure5. 已致敏小鼠在Der f激發後腹腔給予低劑量NAC的小鼠肺臟組織脂質過氧化作用……………………………………………………. 44
Figure6. 已致敏小鼠在Der f激發後腹腔給予低劑量NAC的BAL與血液
中O2-的產量………………………………………………………….. 45
Figure7. 已致敏小鼠在Der f激發後腹腔給予低劑量NAC的BAL與血清
中Nitrite的產生……………………………………………………… 46
Figure8. 已致敏小鼠在Der f激發前與激發後腹腔給予高劑量NAC的肺部發炎反應與血液中噬酸性球數目………………………………… 47
Figure9. 已致敏小鼠在Der f激發前與激發後腹腔給予高劑量NAC的血清中Der f特異性IgG1和IgG2a/2b抗體的產生………………….. 48
Figure10.已致敏小鼠在Der f激發前與激發後腹腔給予高劑量NAC對小鼠BAL total protein的影響………………………………………….. 49
Figure11.已致敏小鼠在Der f激發前與激發後腹腔給予高劑量NAC對
BAL與血清中Nitrite產生的影響………………………………….. 50
Figure12.已致敏小鼠在Der f激發前與激發後腹腔給予高劑量NAC對
肺泡沖洗液中IL-6、TNF-α濃度的影響………………………….. 51
Figure13.已致敏小鼠在Der f激發後的小鼠支氣管過度反應指標(APTI:airway pressure time index)…………………………………………... 52
Figure14.小鼠經Der f激發後不同時間點的肺部發炎反應………….………. 53
Figure15.小鼠經Der f激發後血液中O2-產量增加………………………….. 54
Figure16.小鼠經Der f激發後BAL total protein的產生……………………… 55
Figure17.連續灌食小鼠NAC一週對於Der f激發後12、24小時肺部發
炎反應的影響………………………………………………………… 56
Figure18.連續灌食小鼠NAC一週對於Der f激發後血液中O2-產量的影響.. 57
Figure19.連續灌食小鼠NAC一週對於Der f激發後肺泡沖洗液中IL-6、TNF-α的影響………………………………………………………… 58
Figure20.連續灌食小鼠NAC一週對於Der f激發後肺泡沖洗液中 Nitrite產生的影響………………………………………………………….. 59
Figure21.持續給予NAC對多次氣管內接種Der f小鼠肺部發炎反應的
影響…………………………………………………………………. 60
Figure22.持續給予NAC對多次氣管內接種Der f小鼠BAL IL-5、IL-6、TNF-α產生的影響………………………………………………….. 61
Figure23.持續給予NAC對多次氣管內接種Der f小鼠血液中O2-產量
的影響……………………………………………………………….. 62
Figure24.持續給予NAC對多次氣管內接種Der f小鼠BAL Nitrite產生
的影響……………………………………………………………….. 63
Figure25.持續給予NAC對多次氣管內接種Der f小鼠BAL total protein產生的影響………………………………………………………….. 64
Figure26.持續給予NAC對多次氣管內接種Der f小鼠支氣管過度反應
指標的影響………………………………………………………….. 65
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