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系統識別號 U0026-0812200915240138
論文名稱(中文) 探討陽離子-氯離子共同運輸蛋白在嗜中性白血球活化過程中所扮演的角色
論文名稱(英文) Functional role of cation-chloride cotransporter in neutrophil activation
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 97
學期 2
出版年 98
研究生(中文) 梁峻豪
研究生(英文) Jun-Hao Liang
電子信箱 s2696109@mail.ncku.edu.tw
學號 s2696109
學位類別 碩士
語文別 英文
論文頁數 70頁
口試委員 指導教授-沈孟儒
口試委員-呂增宏
口試委員-謝奇璋
中文關鍵字 陽離子-氯離子共同運輸蛋白  嗜中性白血球  自由基 
英文關鍵字 neutrophil  KCC  ROS  cation-chloride cotransport  K-Cl cotransport  NADPH oxidase 
學科別分類
中文摘要 嗜中性白血球是人類先天免疫中最重要且所佔比例最高的免疫球。先前的研究指出在嗜中性白血球受到刺激會有胞內氯離子外流至吞噬體及胞外的現象,此一氯離子聚集能其和H2O2 反應產生細菌毒殺物質HOCl。最近的研究指出:氫氯離子反向運輸蛋白ClC-7為溶體中最主要的氯離子通透路徑,其中溶體會併入吞噬體中,間接暗示ClC-7在嗜中性白血球在活化過程中扮演的角色。此外,有另一種氯離子通道ClC-3能夠調控NADPH氧化產生。在一臨床研究也發現纖維性囊腫(cystic fibrosis)病人由於一種氯離子運輸蛋白 (CF transmembrane conductance regulator, CFTR)的失能而導致嗜中性白血球的殺菌能力較正常者差。這些結果顯示氯離子運輸蛋白能夠影響NADPH氧化活性以及HOCl產生。在另一方面,研究顯示嗜中性白血球吞噬體的鉀離子內流能促進殺菌酵素活性並增加殺菌能力,而此一媒介鉀離子內流的蛋白並未明朗。以上研究說明氯離子及鉀離子的流動在嗜中性白血球活化過程中扮演重要的角色,然而,目前並沒有研究顯示陽離子-氯離子共同運輸蛋白(包括KCC及NKCC)是否影響嗜中性白血球活化或主導氯、鉀離子通透。
首先我們利用lucigenin冷光監測嗜中性白血球所釋放的自由基來檢視nicotin-amide adenine dinucleotide phosphate (NADPH) oxidase的活性。並利用KCC專一性抑制劑[(dihydroindenyl)oxy] 以及NKCC 專一性抑制劑bumetanide來進行實驗。結果顯示KCC在phorbol 12-myristate 13-acetate (PMA),N-formyl-methionyl-leucyl-phenylalanine (fMLP)以及lipopolysaccrides刺激的嗜中性白血球自由基釋放中扮演了重要的角色。利用反轉錄-聚合連鎖反應以及西方墨點法確認人類嗜中性白血球中四種KCC異構體的訊息核酸及蛋白表現。此外,利用放射性同位素36Cl 及86Rb證明KCC媒介PMA誘導的嗜中性白血球之鉀、氯離子外流現象。我們也同時在小鼠嗜中性白血球以及已分化的類嗜中性白血球細胞株(dHL-60)中證明KCC在PMA或fMLP刺激白血球活化產生自由基的過程中扮演了重要角色。最終利用KCC3基因剔除鼠之嗜中性白血球證明KCC3參與在嗜中性白血球活化過程中。本研究的重要性為發現鉀氯離子共同運輸蛋白KCC在嗜中性白血球活化產生自由基的過程中扮演了重要角色,以及KCC提供產生細菌毒殺物質的鉀、氯離子來源。
英文摘要 Polymorphonuclear neutrophils (PMNs), also referred to as neutrophils, are the most abundant and important type of white blood cells and form an essential part of the immune system. Several studies indicated that there is a dramatic and irreversible efflux of intracellular chloride during neutrophil phagocytosis. This chloride movement leads to high chloride concentration in phagosome or outermembrane microenvironment in order to interact with hydrogen peroxide (H2O2) to produce hypochlorous acid (HOCl) that is the most critical bacterial killing chemical. Some literatures show that the H+/ 2Cl- antiporter chloride channel 7 (ClC-7) is the primary chloride permeation pathway in lysosome, that can fuse with phagosome. ClC-3, another member of ClC family regulates phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants after endotoxin priming. Other studies proved that the neutrophils isolated from cystic fibrosis (CF) patients have poor bacterial killing activity due to dysfunction of CF transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. On the other hand, the K+ influx of the phagosomal membrane makes the vacuole hypertonic and it is suitable for activating granule enzyme. The identity of this protein which can mediate K+ influx is controversial. All above results suggest that the Cl- and K+ transport systems are important for NADPH oxidase activity or HOCl generation. However, it is not clear about the dominant Cl- and K+ permeation pathway for the Cl- and K+ movements during neutrophil activation. So far, all researches focused on the relationship between chloride channels and neutrophil function. This study aims to investigate the role of cation-chloride cotransporter (CCC) including K-Cl cotransport (KCC) and Na-K-Cl cotransport (NKCC) during neutrophil activation. We at first examined the role of cation-chloride cotransporters in NADPH oxidase activation by utilizing different cation-chloride cotransport inhibitors and lucigenin-enhanced chemiluminescence assay. DIOA, the KCC blocker inhibits phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharides-induced NADPH activity of human neutrophils. In contrast, bumetanide, the NKCC blocker does not inhibit this activation. This implies KCC, instead of NKCC plays a functional role in PMA, fMLP or lipopolysaccride-induced NADPH activity of human neutrophils. Western blotting and RT-PCR showed that human neutrophils express all KCC isoforms (KCC1-KCC4). In addition, PMA-induced isotopic 36Cl and 86Rb efflux can be abolished by DIOA. This result indicated KCC mediates ion movements of PMA-induced Cl- and K+ efflux in human neutrophils. Besides, we found that KCC expresses and is important for PMA or fMLP-induced NADPH activity in differentiated neutrophil-like HL-60 cells (dHL-60). Consistently, KCC is necessary for PMA or fMLP-induced NADPH activity in murine neutrophils by chemiluminescence assay. Finally, we found that KCC3 knockout PMNs have poor PMA-induced NADPH oxidase activity compare to wildtype PMNs. That indicated KCC3 participates in the activation. This study indicated that KCC can regulate NADPH oxidase activity and provides K+ and Cl- movements which are necessary for bacterial killing activity of PMNs.
論文目次 Abstract 6

中文摘要 9

Introduction 11

Materials and methods 27

Results 33

Discussion 41

References 45

Figures 56
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