臺灣博碩士論文加值系統

　　顆粒性白血球主要是以產生氧活性中間產物(ROS)來殺死入侵身體的微生物，這些ROS包含了superoxide、H2O2和ONOO-等。嗜伊紅性白血球 (Eosinophil;Eos)為顆粒性球的一員，主要參與第一型的的過敏反應(type I hypersensitivity)與寄生蟲的感染。嗜伊紅性白血球主要存在於某些發炎的上皮組織如呼吸道、腸胃道和泌尿道，其細胞質的顆粒中含有major basic protein、eosinophil-derived neutrotoxin、eosinophil peroxidase等蛋白質，這些蛋白對寄生蟲、腫瘤細胞和宿主細胞而言都是有毒性的，而嗜伊紅性白血球可以產生大量的ROS，甚至多於嗜中性白血球。嗜伊紅性白血球聚積在發炎組織是透過何種機制顯然是很複雜的，這需要許多不同細胞激素和趨化素的產生如IL-3、IL-5，RANTES來達到。近來的研究中指出ROS可以透過改變黏著分子(adhesion molecule)的表現與功能，而調控白血球的運送 (traffic)。
　　為了要研究顆粒性白血球在發炎時，如何調控ROS的產生與細胞黏著，我們製備了一群對抗嗜伊紅性白血球表面分子的單株抗體並進行不同的功能試驗，我們分析了在這些抗體的刺激之下，嗜中性白血球與嗜伊紅性白血球的呼吸爆發 (respiratory burst)是否受到改變。此外，我們也測量這些抗體對於嗜伊紅性白血球的細胞株-HL60-Clone15黏著到VCAM-1與ICAM-1的影響。我們發現一個稱為8-7-D的抗體可以增加ROS的產生，並降低細胞黏著; 另外一個稱為7-1-D的抗體則可透過與ROS產生無關的方式增加細胞黏著，為了要尋找這兩個有功能的抗體可能結合的接受器(receptor)為何，我們將抗體和特殊beads連結(cross-link)做成affinity column，並以此column抓住可與抗體結合的蛋白，此外，我們也進行了免疫沉澱法。在8-7-D抗體的部分，我們發現其可與分子量約80kDa的分子結合，在進行mass spectrometer後，推斷可能是A disintegrin and metalloproteinase family(ADAM)或是CD44，這些研究將有助於釐清嗜伊紅性白血球表面分子，如何調控ROS的產生與細胞黏著。
　　抗嗜中性球抗體 (Antineutrophil cytoplasmic antibody ; ANCA )出現於患有全身性血管炎的病患血清中，ANCA主要作用的兩個自體抗體為proteinase3(PR-3)與myeloperoxidase(MPO)。研究指出ANCA首先透過與嗜中性白血球表面的PR-3或MPO結合，促使嗜中性白血球活化並釋放許多發炎媒介物來致病，這些發炎媒介物如ROS對內皮細胞有很強的毒性，因而產生血管炎。此外，除了以抗體的F(ab’)片段與嗜中性白血球結合外，ANCA亦可以其Fc片段與嗜中性白血球的Fc receptor反應，尤其是Fc�RIIa。
　　我們研究兩位血清中分別含有MPO-ANCA與PR-3 ANCA自體抗體的病人，對於嗜中性白血球活化的影響。首先，我們確定了兩種ANCA都能刺激呼吸爆發，接著，我們也發現ANCA的刺激可以增加嗜中性白血球黏著到ICAM-1與HUVEC，然而，如果先處理catalase將H2O2去除，那麼黏著上升的情況就會消失。這些結果顯示ANCA在嗜中性白血球黏著分子的功能調節上，扮演著角色，這將有助於了解ANCA在致病機轉上的角色。
　　在第一部分的實驗中，我們製造一群對抗嗜伊紅性白血球的單株抗體，並且分析這群抗體在白血球黏著和製造ROS的影響。之後針對兩個具有功能的抗體，尋找其可能結合的分子，我們發現8-7-D抗體辨識的分子有可能為ADAM family或是CD44，之後需要更多的實驗來證明這點。而在第二部分的實驗中，我們則是探討病理性的抗體-ANCA對嗜中性白血球產生ROS與黏著的影響，我們發現ANCA可能藉由增加嗜中性白血球ROS產生，來改變嗜中性白血球的黏著。這些實驗將有助於更了解白血球如何調控ROS產生與黏著分子。

　　The microbicidal activity of granulocytes depends on the generation of reactive oxygen species (ROS) such as H202 and superoxide anion. Eosinophils , a subpopulation of granulocytes, are implicated in immediate hypersensitivity and the responses to parasitic pathogens. Eosinophils are found in certain inflammatory epithelial tissues, such as respiratory, gastrointestinal, and genitourinary tracts. Eosinophils contain specific granules which include major basic proteins, eosinophil- derived neurotoxin, and eosinophil peroxidase, and are potent in producing reactive oxygen species (ROS). All of these are toxic to parasites, tumor cells, and hosts. The mechanisms of eosinophil recruitment to inflammatory sites are apparently complex and include increased production of eosinphils by a variety of factor, such as IL-3, IL-5, and RANTES. Recent investigations suggest that ROS may regulate leukocyte trafficking by modulating adhesion molecules.
　　In order to study the regulation of ROS production and cell adhesion on granulocytes during inflammation, we prepared a panel of monoclonal antibodies against human eosinophils. We first measured the respiratory burst of eosinophils and neutrophils stimulated by these mAbs. The effect of these mAbs on HL60-clone15, a eosinophil-like cell line, adhesion to VCAM-1 and ICAM-1 were also measured. We found that one mAb, 8-7-D, increased ROS production and decreased adhesion. We also found another antibody, 7-1-D, can increase cellular adhesion in a ROS independent manner. In order to identify the specific receptors interacting with these functional antibodies, we made affinity column and perform immunoprecipitation assay. These studies may facilitate the understanding of the eosinophil surface molecules in regulating cell adhesion and ROS production.
　　Autoantibodies may contribute to the pathogenesis of human disease by disrupting normal regulation of ROS production and cellular adhesion. Antineutrophil cytoplasmic antibodies (ANCA) are present in the plasma of patients with systemic vasculitis such as Wegener’s granulomatosis (WG). The 2 major autoantigens for ANCA are proteinase 3(PR-3) and myeloperoxidase(MPO). In vitro, the pathogenicity begins when ANCA binds to the MPO or PR-3 on the surface of neutrophils. Neutrophils are then activated and release inflammatory mediators. e.g. ROS, which is cytotoxicity to endothelial cells and can lead to vasculitis. ANCA induced neutrophil activation involves not only binding of the antibodies via their F(ab’)2-fragments, but also interaction of their Fc-fragments with Fc-receptors on neutrophils, particularly with the Fc�RIIa-receptor. Affinity-purified ANCA can also induce monocytes to produce oxygen radical.
　　We investigate two patients who have MPO-ANCA and PR-3 ANCA respectively. We used these two kinds of antibodies to study the activation of neutrophil. We first confirmed that ANCA can increase neutrophil respirtory burst. We also found that with ANCA stimulation, neutrophil adhesion to ICAM -1 and HUVEC increased. But if we combine catalase in this adhesion experiment, the ANCA-mediated increase is abolished. These results indicate that ANCA play a role in regulating neutrophil adhesion molecules.
　　In the first part, we prepared a panel of monoclonal antibodies against human eosinophils. We measured the respiratory burst and adhesion of granulocytes stimulated by these mAbs. We then identified the specific receptors interacting with two functional antibodies by affinity column and immunoprecipitation assay. We found that 8-7-D may bind to ADAM family or CD44. In the second part, we used ANCA to study the activation of neutrophils. We found that ANCA may increase neutrophil adhesion in a ROS dependent manner. These results may help us to unravel the physiological and pathogenic roles of leukocytes activation in human disease.

中文摘要………………………………… 3
英文摘要………………………………… 5
目錄……………………………………… 8
圖表目錄………………………………… 10
第一章 序論…………………………… 12
第二章 材料與方法……………………… 17
1.細胞株與培養基……………………… 17
2.顆粒性白血球及嗜伊紅性白血球的純化…………… 19
3.單株抗體製備……………………… 20
4.抗體純化……………………… 23
5.SDS-PAGE膠體電泳分析………………… 25
6.白血球黏著反應分析…………………… 28
7.Respiratory burst assay………… 30
8.Immunoprecipitation…………………………… 32
9.抗原的分離…………………… 35
第三章 實驗結果
第一部分 對抗嗜伊紅性白血球表面分子的單株抗體製造與功能分析…… 38
A. Anti-Eos mAbs的細胞表面分子染色………… 38
以Hybridoma方式製造11株Anti-Eos mAbs………… 38
全部的Anti-Eos mAbs都可染Eos表面分子……… 38
8株Anti-Eos mAbs可染經butyric acid和IL-5引導成熟的HL60-clone15 (Eos cell line)………… 39
有些Anti-Eos mAbs也可染其他血球細胞或細胞株表面分子…39
3株Anti-Eos mAbs可染上皮細胞表面分子…… 39
B. Anti-Eos mAbs的功能分析…………………… 40
8-7-D和7-1-D抗體的刺激會影響HL60-clone15的黏著 40
純化的抗體依然可辨識HL60-clone15………… 40
8-7-D抗體可增加嗜伊紅性球，嗜中性球 respiratory burst當使用Flow cytometry方式偵測…… 41
8-7-D抗體可以增加HL60-clone15 respiratory burst 41
8-7-D抗體可以增加Neu respiratory burst當使Chemiluminescence method方式偵測… 41
8-7-D和7-1-D抗體的刺激不影響HL60-clone15表面total VLA-4的表現…42
C. 以免疫沉澱法和antibody cross-linked column可觀察到與抗體反應的細胞表面抗原… 43
第二部分: 抗嗜中性球抗體(ANCA)對嗜中性球活化的影響…………………… 46
A. 以ELISA確定病人血液中含有何種ANCA… 46
B. MPO-ANCA和PR3-ANCA皆可增加嗜中性球呼吸爆發… 46
C. MPO-ANCA可增加嗜中性球與ICAM-1和HUVEC的黏著…47
D. Catalase可阻斷由ANCA所引起的嗜中性球黏著增加…………………… 47
第四章 討論…………………49
Reference list………………76








圖 表 目 錄

Table 1. 利用FACES的方式分析anti-Eos mAb與不同細胞結合的情形.. 55
圖1. 以FACS的方式分析anti-Eos mAb與人類Eosinophil結合的情形 56
圖2. .以FACS的方式分析anti-Eos mAb與HL60-clone15結合的情形.. 57
圖3. 某些anti-Eos mAb會影響HL60-clone15與VCAM-1的黏著 58
圖4. 某些anti-Eos mAb會影響HL60-clone15與ICAM-1的黏著 59
圖5. 利用SDS-PAGE分析自小鼠腹水中純化的抗體 60
圖6. 以FACS的方式分析純化的anti-Eos mAb 8-7-D,7-1-D與HL60-clone15結合的情形 61
圖7. (a)以FACS的方式分析anti-Eos mAb刺激對Eosinophil產生ROS的影響
(b)對Neutrophil產生ROS的影響 62
圖8. (a)以Florescence reader直接偵測anti-Eos mAb的刺激對HL60-clone15產生
ROS的影響 (b)純化的8-7-D抗體對HL60-clone15產生ROS的影響 63
圖9. 以化學冷光法的方式分析anti-Eos mAb supernatant 8-7-D對granulocyte產生ROS的影響 64
圖10. 純化的8-7-D抗體會降低HL60-clone15對於VCAM-1的結合能力 65
圖11. 純化的7-1-D抗體會增加HL60-clone15對於VCAM-1的結合能力 66
圖12. 利用FACS的方式分析HL60-clone15以抗體刺激後細胞表面total VLA-4表現的情形 67
圖13. 利用免疫沉澱法分析抗體所辨識的細胞表面分子 68
圖14. 利用antibody cross-linked column分析抗體所辨識的細胞表面分子 70
圖15. 利用MPO-ELISA assay分析兩個ANCA病人血清中MPO antibody的 71
含量
圖16. MPO-ANCA和PR3-ANCA皆能刺激嗜中性球呼吸爆發 72
圖17. MPO-ANCA的刺激對嗜中型球與ICAM-1黏著的影響 73

圖18. MPO-ANCA的刺激對嗜中型球與HUVECs黏著的影響 74
圖19. Catalase可阻斷因MPO-ANCA而造成的嗜中性球與HUVECs黏著上 75
升的情形

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