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系統識別號 U0026-1207201816484200
論文名稱(中文) 探討小鼠血清誘發型免疫關節炎嗜中性白血球細胞外網狀結構的形成與IL-1β生成處理的氧化還原調控
論文名稱(英文) Redox Regulation of Neutrophil Extracellular Traps Formation and IL-1β Processing in Serum-induced Arthritis
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 106
學期 2
出版年 107
研究生(中文) 許正綱
研究生(英文) George C. Hsu
學號 S96034027
學位類別 碩士
語文別 英文
論文頁數 39頁
口試委員 指導教授-謝奇璋
口試委員-陳芃潔
口試委員-莊國賓
中文關鍵字 活性含氧物  嗜中性球胞外網狀結構  類風濕性關節炎  K/BxN 血清誘發關節炎  IL-1β 
英文關鍵字 ROS  NET  Rheumatoid arthritis  IL-1β  K/BxN serum-induced arthritis 
學科別分類
中文摘要 嗜中性球胞外網狀結構 (neutrophil extracellular traps, NETs)主要是由嗜中性白血球在遇到特殊刺激時所進行的一種細胞凋亡機制,整個過程被稱為NETosis。NETosis的過程中,會釋放DNA、組織蛋白、蛋白酶、自體抗原與活性含氧物 (reactive oxygen species, ROS)等,而導致鄰近組織的損傷和發炎。然而,一些研究指出與NETs一起被釋放的絲氨酸蛋白酶 (serine proteases)能夠降解促發炎介質並隨後消除發炎症狀。在我們之前的研究中,我們以類風濕性關節炎(rheumatoid arthritis, RA) 的動物模式―血清誘發型關節炎(serum-induced arthritis)為材料,發現ROS會通過抑制負責IL-1β成熟的組織蛋白酶B (cathepsin B)而降低發炎反應的產生。另外目前的研究顯示,ROS的產生對NET的形成至關重要。因此,我們推測在無法產生ROS的Ncf1-/-缺陷小鼠,之所以會有嚴重的關節炎症狀,是由於ROS產生不足而無法形成NETs,進而無法利用隨NET釋放的蛋白酶去降解促炎細胞因子(pro-inflammatory cytokines)。因此,本研究著手探討ROS、自體免疫性關節炎與NET加工處理細胞因子的關係。
在這項研究中,我們使用會導致關節炎的K/BxN小鼠血清去誘發野生型小鼠和Ncf1-/-缺陷小鼠產生關節炎,並且使用非侵入性的IVIS Spectrum、micro-CT和物理測量來監測發炎的嚴重程度。此外,為了研究K/BxN血清是否可誘導NETosis,我們將嗜中性球細胞與K / BxN血清和PMA (phorbol 12-myristate 13-acetate) 一起培養以誘導NETs形成。除了識別NETosis外,我們還試圖建立一種可以分離NETs並保留蛋白酶活性和生物學功能的純化方式,以利日後研究使用。
結果顯示,我們無法在發炎較嚴重的Ncf1-/-缺陷小鼠偵測到ROS的訊號,同時Ncf1-/-缺陷小鼠在關節腫脹程度、骨頭侵蝕與骨密度流失都比野生型小鼠來的嚴重。這些數據顯示ROS可能在血清誘導的關節炎中扮演抑制發炎的角色。在NET的部份,我們用螢光顯微鏡揭示了K / BxN血清能夠促使NET 的產生。最後,我們成功發展並證明NET片段可以從嗜中性白血球細胞與NET的混合網中分離出來(neutrophil-NET mesh),同時保留其蛋白酶活性和處理pro-IL-1β將其轉化為IL-1β的功能。
總之,我們的實驗結果連接了NET,ROS和RA之間的關聯。缺乏ROS可能導致NET形成不足並導致發炎無法得到控制。儘管NET分離方法複雜且在各實驗室間有所不同,但我們成功地建立了可以產生足夠量NET的分離方法。本研究對於日後在關於類風濕性關節炎的致病機制與NET相關疾病的研究上,希望能在實驗設計與的疾病控制策略上有所貢獻。
英文摘要 Neutrophil extracellular traps (NETs) are structures of chromatin filaments decorated with histones, proteases and granular proteins. The release of NETs, also known as NETosis, expose DAMP signals, autoantigens, proteases and reactive oxygen species (ROS) to the extracellular milieu and often lead to tissue damage and inflammation. However, it is also reported that serine protease released along with NETs are capable of degrading pro-inflammatory mediators and subsequently resolve inflammation. In our previous studies, we discovered that ROS regulate serum-induced arthritis, a murine model for immune-mediated arthritis, including rheumatoid arthritis (RA). ROS regulate arthritic inflammation via suppressing cathepsin B, which mediates IL-1β maturation in the joints. Moreover, the production of ROS is critical for NET formation. As a result, we postulated that the development of severe joint inflammation in ROS-deficient mice is due to insufficient generation of ROS and NETs, which revoke the degradation of pro-inflammatory cytokines by NET-derived proteases. Hence, we set out to investigate ROS regulations in NET-mediated cytokine processing in autoimmune arthritis.
In this study, we treated wild-type mice and ROS-deficient mice Ncf1-/- with K/BxN arthritogenic mice serum. Disease severity was monitored with IVIS Spectrum, micro-CT and physical measurements. In order to investigate whether K/BxN serum can induce NETosis, neutrophils were incubated with K/BxN serum and phorbol-12-myristate-13-acetate (PMA) to induce NETs formation. In addition to identify NETosis, we also conducted experiments to establish a feasible approach to isolate NETs while preserving the protease activity and biological function.
Our results showed that even though ROS signals were only seen in wild-type mice, joint swelling was significantly more severe in Ncf1-/- mice compared with wild-type controls. Moreover, significant bone erosion and bone mineral density loss were found in Ncf1-/- mice when compared with those in wild-type mice. These data support the hypothesis that ROS is anti-inflammatory in serum-induced arthritis. We also showed with fluorescence microscopy that large amount of extracellular DNA stretches were generated after K/BxN serum and PMA were added. In the final part, we demonstrated that NET fragments can be isolated from the neutrophil-NET meshes while retain its protease activity and biological function. Furthermore, NET-derived neutrophil elastase was active in processing pro-IL-1β to the mature form.
In conclusion, our data gives awareness of the association between NETs, ROS and RA. Lacking ROS may lead to insufficient NET formation and cause deteriorating inflammation. Even though the methodology of NET isolation have been proved to be tricky, we successfully establish an approach which yields sufficient amount of viable NET sample, which will be applied to the future experiments regarding the mechanism of rheumatoid arthritis and may aid the design of effective strategies to control NET-associated diseases.
論文目次 Abstract..............................................III
中文摘要 ...............................................IV
ACKNOWLEDGEMENT.........................................V
Chapter 1: Introduction.................................1
1.1 Rheumatoid arthritis................................2
1.2 Reactive oxygen species and the NADPH oxidase complex ........................................................2
1.3 The tool to investigate early RA onset: the K/BxN animal model............................................3
1.4 Brief introduction of NETs..........................4
1.5 NETosis.............................................5
1.6 The beneficial effects of NETs......................6
1.7 Research objective..................................7
Chapter 2: Materials and Methods........................8
2.1 Mice................................................9
2.2 Serum-induced arthritis.............................9
2.3 In vivo imaging of ROS.............................10
2.4 In vivo micro-CT analysis and 3D model reconstruction .......................................................10
2.5 Neutrophil isolation...............................11
2.6 NET induction and isolation........................11
2.7 Immunofluorescent staining of NETs.................12
2.8 In vitro quantification of NET proteins and DNA components.............................................12
2.9 Analysis of NET-associate protease activity........13
2.10 IL-1β bioassay and immunostaining analysis of NF-κB nuclear translocation..................................13
2.11 Statistical analysis..............................14
Chapter 3: Results.....................................15
3.1 ROS play an anti-inflammatory role in serum-induced arthritis..............................................16
3.2 K/BxN serum promotes PMA-induced NET formation.....16
3.3 NET fragments can be isolated from neutrophil-NET complexes while retain its protease activity and biological function....................................17
Chapter 4: Discussion..................................20
Chapter 5: Figures and Legends.........................24
Fig. 1.................................................25
ROS play an anti-inflammatory role in serum-induced arthritis..............................................25
Fig. 2.................................................26
Ncf1- / - mice developed severe bone erosion and density lost...................................................26
Fig. 3.................................................27
Flow chart for PMA-induced NETs formation model and NET isolation..............................................27
Fig. 4.................................................28
PMA-induced NET formation..............................28
Fig. 5.................................................29
K/BxN serum promotes PMA-induced NET formation.........29
Fig. 6.................................................30
NET fragments can be isolated from neutrophil-NET complexes..............................................30
Fig. 7.................................................31
Isolated NET sample retains its protease activity...............................................31
Fig. 8.................................................32
Isolated NETs maintain its biological activity in promoting functional IL-1β.............................32
Fig. 9.................................................33
Summary figure.........................................33
Chapter 6: References..................................34
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