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研究生:黃思偉
研究生(外文):Szu-Wei Huang
論文名稱:外源性介白素-6、介白素-13和丙型干擾素加劇腸病毒71型感染小鼠之肺臟異常
論文名稱(外文):Exogenous interleukin-6, interleukin-13 andinterferon-gamma exacerbate pulmonaryabnormality in enterovirus 71-infected mice
指導教授:余俊強
指導教授(外文):Chun-Keung Yu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微生物暨免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:52
中文關鍵詞:腸病毒71型神經性肺水腫細胞素風暴
外文關鍵詞:EV71neurogenic pulmonary edemacytokine storm
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臨床上發現,中樞神經與全身性的炎症反應可能是感染腸病毒71 型 (EV71)
後導致神經性肺水腫 (neurogenic pulmonary edema;NPE) 的重要因子。小鼠適應
株EV71/MP4 顱內感染7 日齡ICR 小鼠,在中樞神經可發現無菌性腦膜炎和腦幹
腦炎,並伴隨血清中與NPE 相關之細胞素包括IL-6、IL-10、IL-13 和IFN-γ濃度
暫時性升高;同時EV71 感染小鼠出現後肢癱瘓以及肺功能異常,組織病理學檢
查也發現EV71 感染小鼠死亡前二天發生肺氣腫,但卻沒發生肺水腫。因此,我
們假設EV71 感染中樞神經,伴隨血清中足夠濃度的IL-6、IL-13 和IFN-γ才能引
發NPE。首先,我們給予EV71 感染小鼠腹腔注射IL-6、IL-13 和IFN-γ,與控制
組相比,小鼠在死亡前出現嚴重肺功能異常,及重度肺氣腫和輕微肺水腫,並且
小鼠肺重與體重比顯著增加;只給予EV71 感染小鼠IL-6 與IFN-γ或單獨IL-13
無法得到相同的結果,顯示IL-6、IL-13 和IFN-γ的共同作用在EV71 引起NPE 扮
演重要角色。接著,為了釐清 EV71 感染中樞神經合併細胞素所導致肺部機能惡
化是否為EV71 特有的現象,我們給予第一型單純皰疹病毒 (herpes simplex
virus-1; HSV-1) 感染小鼠腹腔注射IL-6、IL-13 和IFN-γ,小鼠雖出現腦炎和神經
症狀,但沒有肺臟異常加劇或發生NPE。總括而言,本研究證明以外源性IL-6、
IL-13 和IFN-γ注射EV71 感染小鼠可加劇肺臟異常、促進神經性肺水腫發生,且
為EV71 感染所特有的現象。
Clinical observations suggest that both systemic and CNS inflammatory responses
are involved in the induction of neurogenic pulmonary edema (NPE) in enterovirus 71
(EV71) patients. In addition, aseptic meningitis and brainstem encephalitis were noted
in the CNS accompanying with a transient increase of PE-associated cytokines in
serum including IL-6, IL-10, IL-13 and IFN-γ. In an EV71 experimental infectious
model in which 7-day-old mice were intracranially inoculated with a mouse-adapted
EV71 strain MP4, we observed that the animals developed hind-limb paralysis and
pulmonary dysfunction (a marked decrease in peak inspiratory flow, peak of expiratory
flow, minute volume and tidal volume) and emphysema without evidence of NPE a
few days before death. We assumed that the development of EV71-induced NPE may
require a combined effect of CNS infection and a sufficient amount of IL-6, IL-13, and
IFN-γ. We found that EV71-infected mice with post-treatment of IL-6, IL-13, and
IFN-γ developed mild PE and severe emphysema accompanying with a more severe
pulmonary dysfunction than control mice. Post-treatment of EV71-infected mice with
IL-6 and IFN-γ or IL-13 only showed no such effect. Furthermore, in order to check if
this combined effect is specific to EV71-induced encephalitis, we also treated herpes
simplex virus-1 (HSV-1) infected mice with IL-6, IL-13, and IFN-γ, but no such effect
was found. In conclusion, we proved that PE could be induced in EV71-, but not
HSV-1-infected mice with exogenous IL-6, IL-13, and IFN-γ treatment.
中文摘要.....................................................................................................................I
英文摘要................................................................................................................... II
誌謝....………………………………………………………………………………………………………….………III
目錄..........................................................................................................................IV
圖表目錄...………………………………………………….…………………………..……………….VII
第一章 緒論.............................................................................................................. 1
(一)腸病毒71 型之病毒學概論與流行病學........................................................ 1
(二)腸病毒71 型之致病機轉.................................................................................. 2
(三) 腸病毒71 型之中樞神經侵犯併發神經性肺水腫..................................... 3
(a) 血液動力學機轉 (hemodynamic mechanism) ................................. 4
(b) 炎症性機轉 (inflammatory mechanism).......................................... 5
(四) 腸病毒71 型感染的動物模式........................................................................ 6
(五) 單純皰疹病毒 (Herpes simplex virus; HSV) 感染的小鼠腦炎模式... 7
第二章 研究目的與特異目標.................................................................................. 8
第三章 材料與方法................................................................................................ 10
A 材料....................................................................................................................... 10
A-1 實驗動物........................................................................................... 10
A-2 細胞株............................................................................................... 10
A-3 病毒................................................................................................... 10
A-4 藥品................................................................................................... 11
A-5 抗體與試劑組................................................................................... 12
A-6 耗材................................................................................................... 12
A-7 儀器................................................................................................... 13
B 方法....................................................................................................................... 14
B-1 細胞培養........................................................................................... 14
B-2 病毒培養與感染............................................................................... 15
B-3 組織切片與染色觀察....................................................................... 17
B-4 Sandwich ELISA:血清及臟器細胞素監測.................................. 18
B-5 肺功能測定:Unstrained Whole Body Plethysmography ........... 20
B-6 肺重與體重比量測........................................................................... 20
B-7 統計方法........................................................................................... 21
C 實驗設計.............................................................................................................. 21
C-1 小鼠感染腸病毒71 型的肺部組織病理和臟器及血清細胞素變化
................................................................................................................... 21
C-2 給予腸病毒71 型感染小鼠外源性細胞素..................................... 21
C-3 給予第一型皰疹病毒感染小鼠外源性細胞素............................... 22
第四章 結果............................................................................................................ 23
(A) 以顱內注射EV71 的方式感染會導致肺功能下降、中度肺氣腫和肺鬱
血, 而沒有肺水腫的發生。............................................................................... 23
(B) 7 日齡小鼠顱內感染EV71,血清中IL-6、IL-10 和IFN-γ皆有升高的
趨勢。
(C) 給予EV71 感染小鼠IL-6、IL-13 和IFN-γ加劇肺功能異常、顯著增加
肺重與體重比,並且在組織病理學檢查中可見輕微肺水腫及中度至重度肺
氣腫。........................................................................................................................ 24
(D) 只給予EV71 感染小鼠IL-6 和IFN-γ不加劇肺功能之異常,但顯著增
加肺重與體重比,組織病理學檢查可見中度至重度肺氣腫,但不見肺水
腫。............................................................................................................................ 25
(E) 只給予EV71 感染小鼠IL-13 可加劇肺功能異常,卻不增加肺重與體
重比,組織病理學檢查除中度至重度肺氣腫也不見肺水腫。.................... 26
(F) 於EV71 感染後第三天至第四天內3 次給予小鼠IL-6、IL-13 和IFN-γ,
其肺功能變化、肺重與體重比和組織病理學檢查,皆與先前實驗之結果類似。............................................................................................................................ 26
(G) 第一型單純皰疹病毒顱內感染合併IL-6、IL-13 和IFN-γ注射不加劇
小鼠肺功能異常,也不增加肺重與體重比,且組織病理學檢查不見肺水
腫。............................................................................................................................ 27
第五章 討論............................................................................................................ 29
第六章 參考文獻.................................................................................................... 35
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