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研究生： 張明雯
Ming-Wen Chang
論文名稱： 自然殺手細胞經NKG2D/NCR訊息傳遞路徑直接毒殺Mycobacterium kansasii
Direct killing of Mycobacterium kansasii by NK cells via NKG2D / NCR-dependent signaling pathway
指導教授： 宋瑞珍
Ruey-Jen Sung
口試委員:
學位類別： 碩士
Master
系所名稱： 生醫理工學院 - 生命科學系
Department of Life Science
畢業學年度： 99
語文別： 英文
論文頁數： 63
中文關鍵詞： 訊息傳遞路徑 、堪薩斯分枝桿菌 、自然殺手細胞
外文關鍵詞： signaling pathway, Mycobacterium kansasii, Natural killer cells
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    • 自然殺手細胞在宿主先天免疫防禦系統中，扮演了很重要的角色。目前已知其主要的功能為殺死腫瘤細胞或被病毒感染的宿主細胞。然而，自然殺手細胞是否能直接殺死微生物仍然所知甚少。以往的研究顯示，自然殺手細胞可以使用細胞毒性蛋白，例如穿孔素和顆粒溶素直接殺死新型隱球菌-一個致病的酵母菌。因為堪薩斯分枝桿菌所造成的慢性肺部感染與結核分枝桿菌所造成的肺結核相似，本研究中，利用此菌作為研究對象，我探討並發現人類自然殺手細胞株，NK92細胞具有殺死致病細菌-堪薩斯分枝桿菌之現象。進一步研究其毒殺的機制得知，此致死作用是藉由NK92細胞與堪薩斯分枝桿菌的直接接觸所造成。穿孔素和顆粒溶素在其中扮演重要角色，且其中作用的機制是通過活化NKG2D/NCRs, ERK，JNK及p38 MAPK的訊息傳遞路徑所進行。

    Natural killer (NK) cells play a pivotal role in the effector arm of host innate immunity defense system. The major functions of NK cells in killing tumor cells or virus-infected host cells were well established. However, whether NK cells can directly kill microbes remains poorly understood. Previous studies have shown that NK cells can use the cytolytic proteins, perforin and granulysin to directly kill the yeast pathogen Cryptococcus neoformans. In this study, using the pathogenic mycobacterium Mycobacterium kansasii which causes a chronic pulmonary infection resembling pulmonary tuberculosis as the study model, I showed that human NK-92 cells directly kill mycobacterium. Killing of M. kansasii by NK92 cells is contact dependent. NK cells use the cytolytic proteins, perforin and granulysin for direct killing, and the underlying killing mechanism is through NKG2D/NCRs, ERK, JNK and p38 MAPK signaling pathways.

    Table of Contents 中文摘要 i Abstract ii Acknowledgements iii Table of Contents iv List of Figures vi List of Tables viii Chapter 1 Introduction 1 1-1 Non-tuberculous mycobacteria 1 1-1.1 Mycobacterium kansasii 2 1-2 The immune system 2 1-3 NK cells biology 3 1-3.1 Activating and inhibitory receptors of NK cells 4 1-3.2 Signalings controlling the NK cells cytotoxicity 6 1-3.3 Cytotoxic proteins of NK cells 6 1-4 Specific aims 7 Chapter 2 Materials and Methods 9 2-1 Cell line and cell culture 9 2-2 Preparation of M. kansasii 9 2-3 Chemical reagents 10 2-4 Antibodies 10 2-5 RNA extraction and reverse transcription (RT)-PCR 10 2-6 Preparation of protein lysate and immunoblot analysis 12 2-7 Transwell assay 12 2-8 Knock-down of mRNA by siRNA and siRNA transfection 13 2-9 M. kansasii killing by NK92 cells 13 Chapter 3 Results 15 3-1 NK92 cells possess anti- M. kansasii activity 15 3-2 NK92 cells kill M. kansasii and direct contact is essential 15 3-3 NK92 cells requires cytotoxic granules to kill M. kansasii 17 3-4 M. kansasii enhances NKG2D, NKp44 and NKp46 expression 18 3-5 Inhibition of MAP Kinases activity reduces perforin and granulysin production and killing efficacy 18 3-6 Perforin involves killing of M. kansasii 19 3.7 Granulysin also involved M. kansasii killing 20 Chapter 4 Discussion 21 Chapter 5 Reference 25

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