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研究生:郭昱毅
研究生(外文):Yu-Yi Kuo
論文名稱:探討奈米氧化鋅對於清除不可分型流行性感冒嗜血桿菌感染的作用機制在體內與體外試驗
論文名稱(外文):Study on the effects of zinc oxide nanoparticles on ontypeable Haemophilus influenzae clearance in vitro and in vivo
指導教授:李珍珍李珍珍引用關係
指導教授(外文):Chen-Chen Lee
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:基礎醫學研究所碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:42
中文關鍵詞:奈米粒子氧化鋅噬血桿菌巨噬細胞
外文關鍵詞:Haemophilus influenzaenanoparticlesmacrophagezinc oxid
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在我們生活的環境中到處都充斥著含有奈米粒子的相關產品包含在
食、衣、住、行甚至是醫療器材與用品,大部分的奈米相關產品皆是與殺菌
有關,奈米氧化鋅因為具有非常良好的物理性抗紫外線功能,被大量地使用
在化妝品與防曬產品中,由於奈米粒子的粒子大小非常的小,可以輕易的穿
過細胞膜進入到細胞中,因而產生潛在的風險。不可分型流行性感冒嗜血桿
菌 (nontypeable Haemophilus influenzae, NTHi)是一種革蘭氏陰性菌經常潛
伏在人類上呼吸道中造成伺機性的感染,對於小孩、老人和免疫缺失的人較
具有威脅性。在近年來的研究中,越來越多證據顯示奈米粒子對於人體有嚴
重的影響,在本篇研究我們利用細胞與動物實驗來評估關觀察暴露奈米粒子
對於呼吸道感染性疾病的影響。在我們的研究結果中發現,小鼠暴露在 140
μg/ml 奈米氧化鋅環境中並不會直接造成發炎反應,以 NTHi 感染後發炎反
應明顯被誘導上升,暴露奈米粒子與否並不改變發炎反應的程度,但暴露奈
米氧化鋅明顯的降低對於 NTHi 的清除能力、抑制巨噬細胞的活化和減少誘
導性一氧化氮合成酶表現下降。除此之外,在細胞實驗中也發現,將 RAW
264.7 細胞處理 10 μg/ml 奈米鋅後,會增加細胞外 NTHi 的存活率,並抑制
一氧化氮的產生。在本研究中我們發現奈米氧化鋅會抑制巨噬細胞的活化並
影響一氧化氮的生成,進而影響免疫細胞對於外來病原菌的清除。

Nanoparticles (NPs) were widely employed in our living environments. Among
those NPs, zinc oxide nanoparticles (ZnONPs) were commonly used in cosmetics
because of the excellent protective effect to ultraviolet. Moreover, ZnONPs also have
been used in numerous fields to prevent microbial growth. Nontypeable Haemophilus
influenzae (NTHi) is an extracellular Gram-negative and coccobacilli bacteria lacking
of capsule that frequently colonizes human respiratory mucosa and often produces
localized respiratory tract diseases. Recently, several reports indicated inhalation of
ZnONPs may be a risk factor for human health. But only few researches discussed the
relationship between NPs and immune responses. In this study, we used in vitro and
in vivo platforms to assess the potential effect of NTHi clearance after exposure with
ZnONPs. Our data showed that after infection of mice with 1×10
7
NTHi, which
survived in bronchial alveolar lavage fluids (BALF) and lung tissues, and the
inflammatory cells were infiltrated in lung. After exposure with 140 μg/ml ZnONPs
and ZnOMPs for 14 days, we found that the total numbers of inflammatory cells in
BALF and lung did not show obviously change compared to only infection with
NTHi group. However, our data showed that the numbers of active macrophages and
inducible nitric oxide synthase (iNOS) expression were significantly decreased in
BALF. In addition, we found that 10 μg/ml ZnONPs reduced macrophage
extracellular clearance of NTHi and inhibited nitric oxide production in RAW264.7
cells. Our findings reveal that ZnONPs impair NO production and macrophage
activation which may lead to reduce the NTHi clearance.

Abstract ......................................................................................................... IV
中文摘要(Abstract in Chinese) ........................................................................ V
Introduction ...................................................................................................... 1
Development of nanotechnology ........................................................................ 1
The effects of nanoparticles in human ................................................................ 2
Nontypeable Haemophilus influenzae (NTHi) .................................................... 3
Motivation and purpose of this study ................................................................. 4
Materials and methods ..................................................................................... 5
In vitro test of macrophage’s function for treatment with ZnONPs ..................... 5
Chemicals .................................................................................................. 5
Cell culture ................................................................................................ 5
Bacterial culture ......................................................................................... 5
Cell viability assay ..................................................................................... 6
Bactericidal activity ................................................................................... 6
Evaluation of extracellular pathogen .......................................................... 6
Adhesion and phagocytosis assays ............................................................. 7
Nitric oxide (NO) production ..................................................................... 7
RNA extraction .......................................................................................... 8
Reverse transcription .................................................................................. 8
Quantitative real-time polymerase chain reaction (Q-PCR) ........................ 9
Enzyme-linked immunosorbent assay (ELISA) .......................................... 9
Western blot ............................................................................................... 9
In vivo test of macrophage’s function fro treatment with ZnONPs ..................... 11
Animal model ........................................................................................... 11
Broncho-alveolar lavage fluids (BALF) .................................................... 11
Lung .........................................................................................................12
Statistical analysis .....................................................................................14
Results ..........................................................................................................15
Discussion .........................................................................................................20
Tables ..........................................................................................................23
Figures ..........................................................................................................32
References ........................................................................................................23

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