多型核嗜中性白血球(簡稱嗜中性白血球)的有效活化在宿主的防禦上扮演重要的角色。然而，過度的白血球浸潤會導致組織產生非預期性的傷害進而導致疾病的產生。介白素８是一種ELR-CXC趨化素，透過與細胞上特定的穿膜受體CXCR1與CXCR2結合，進而介導白血球的趨化與活化。在先前的研究中，我們透過對介白素8結構特性的分析，開發了一個高效能ELR‐CXC 趨化素受體拮抗劑，稱為G31P-IP10。此篇研究以G31P-IP10為模板，進行N-loop的胺基酸置換，發展出三個具有潛在ELR‐CXC 趨化素受體拮抗劑活性的G31P-IP10類源物(亦為ELR‐CXC趨化素類源物)。我們檢測了這三個G31P-IP10類源物對介白素8刺激下之嗜中性白血球的抗發炎反應效果，發現其中一個G31P-IP10類源物增強了原先G31P-IP10對於ELR‐CXC趨化素受體的拮抗能力。這個G31P-IP10類源物顯著性地抑制了介白素8所誘導的嗜中性白血球趨化作用，彈性蛋白酶(elastase)的釋放，超氧化物質(ROS)的產生以及基質金屬蛋白酶-9(MMP-9)的分泌。結構上的分析也顯示了其在二級結構上與介白素8和G31P-IP10相比沒有明顯的差異。在未來，此G31P-IP10類源物可望能發展為一個有效的抗發炎藥物治療方法。

Polymorphonuclear neutrophils (PMN) activation is important in human host defense. However, excessive neutrophil infiltration in tissue may cause unexpected tissue damage and development of several diseases. Interlukin-8 (IL-8 or CXCL8), an ELR-CXC chemokine, plays a critical role in neutrophil recruitment and activation through binding to the two seven transmembrane G protein–coupled receptors, CXCR1 and CXCR2. Our previous study has provided a new antagonist, G31P-IP10, for ELR-CXC chemokine receptor based on the structure of IL-8. In this study, we employed G31P-IP10 as a template to further develop three G31P-IP10 analogues by site directed mutagenesis. The results demonstrate that one of the G31P-IP10 analogues displays the greatest anti-inflammatory effect and enhanced the antagonism for IL-8 and its receptors. This G31P-IP10 analogue significantly inhibited IL-8/CXCR1/2 triggered neutrophil chemotaxis, elastase release, reactive oxygen species (ROS) response and matrix metallprotease-9 (MMP-9) secretion. Structure analysis showed that the G31P-IP10 analogue shared the identical secondary structure with IL-8 and G31P-IP10.
This G31P-IP10 analogue provides new opportunities in the development of anti- inflammatory therapeutics.

Content
誌謝 (Acknowledgement) ………………………………………….…………….... l
中文摘要........................................................................................................... II
Abstract ………………………………………………………………….…………. III
Content .......................................................................................................... IV
List of figures ................................................................................................... V
List of tables .................................................................................................. VI
Chapter 1 Introduction ..................................................................................... 1
1.1 Neutrophil infiltration .............................................................................. 1
1.2 ELR-CXC chemokines……...……….…………………………………....... 1
1.3 The ELR-CXC chemokines receptors ………….………………………… 3
1.4 The role of interleukin-8 (IL-8) in acute and chronic inflammation……...4
1.5 The structure of interleukin-8 ……………………………………………… 6
1.6 Design of the ELR-CXC chemokine receptor antagonist....................... 7
Chapter 2 Materials and Methods …………..………………………………….. 10
2.1 Chemicals and reagents ………………………………………………..… 10
2.2 Construction of recombinant expressed plasmids …………………….. 10
2.3 Expression of recombinant proteins .................................................... 12
2.4 Expression and purification of recombinant 15N-labelled proteins ....... 13
2.5 Purification of recombinant proteins .................................................... 14
2.6 Characterization of proteins by mass spectrometry…..……………….. 15
2.7 Polymorphonuclear neutrophils (PMN) isolation .................................. 15
2.8 Polymorphonuclear neutrophils (PMN) chemotaxis assay ................... 16
2.9 Elastase activity.................................................................................... 17
2.10 Reactive Oxygen Species (ROS) response assay ............................. 18
2.11 Matrix metalloproteinase9 (MMP-9) activity by zymographpy……..... 19
2.12 Circular Dichroism (CD) spectroscopy ………………………………… 20
2.13 Nuclear magnetic resonance (NMR) spectroscopy ………………….. 20
2.14 Statistical analysis ……………………………………………………….. 21
Chapter 3 Results .......................................................................................... 22
3.1 Construction of recombinant expressed plasmids................................ 22
3.2 Expression and purification of recombinant proteins ........................... 22
3.3 IP10-T12S may be a potential antagonist for IL-8 receptors ............... 24
3.4 IP10-T12S can significantly inhibit IL-8-induced PMN recruitment and chemotaxis ............................................................................................ 24
3.5 IP10-T12S reduced IL-8 induced neutrophil elastase release in dose- dependent ............................................................................................. 25
3.6 IP10-T12S effectively decreased IL-8 stimulated PMN reactive oxygen species (ROS) response ……………………………………………………26
3.7 IP10-T12S decreased matrix metalloproteinase-9 (MMP-9) release from IL-8 stimulated HL-60 cells …………………………………………………27
3.8 IP10-T12S presents the similar secondary structure as G31P-IP10 and IL-8 ……………………………………………………………………………28
3.9 Heteronuclear Single Quantum Coherence (HSQC) NMR …………….28
Chapter 4 Discussion .................................................................................... 30
Chapter 5 Conclusion .................................................................................... 37
References .................................................................................................... 67

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