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研究生:楊文傑
研究生(外文):Wen-Je Yang
論文名稱:豬嗜中性球對各種炎症刺激物之趨化性與氧爆作用
論文名稱(外文):CHEMOTAXIS AND RESPIRATORY BURST OF PORCINE NEUTROPHILS IN RESPONSE TO VARIOUS INFLAMMATORY STIMULANTS
指導教授:王汎熒
指導教授(外文):Fun-In Wang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:英文
論文頁數:130
中文關鍵詞:豬嗜中性球炎症刺激物趨化性氧爆作用假性狂犬病病毒嗜中性球功能抑制放線桿菌胸膜肺炎
外文關鍵詞:Porcine NeutrophilsInflammatory StimulantsChemotaxisRespiratory BurstPseudorabies VirusNeutrophil dysfunctionActinobacillus pleuropneumoniae
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企業化養豬常伴隨許多特定病毒性疾病如假性狂犬病、豬瘟、豬生殖與呼吸綜合症等嚴重蔓延,並引發二次性細菌感染症增多,常見如Actinobacillus pleuropneumoniae (AP), Pasteurella multocida (PM), 和Salmonella choleraesuis。 針對臨床上二次性細菌感染症增多的原因,所提出的假說之一為病毒造成嗜中性球的功能障礙進而影響宿主抵抗病原菌入侵的能力。 嗜中性球最快到達病灶區,殺菌力最強,為個體抵禦細菌感染的主要白血球。 許多人類醫學文獻也證實病毒性疾病在臨床上伴隨細菌感染率的上昇與嗜中性球功能如趨化作用、吞噬作用、氧爆作用、殺菌作用被抑制有關。 本研究希望以假性狂犬病病毒 (Pseudorabies Virus; PrV) 為模式,探討病毒是否抑制豬嗜中性球之防禦功能。 因此,目的之一、希望建立豬嗜中性球次活體 (ex vivo) 之趨化作用與氧爆作用的測試方法(文獻中豬嗜中性球甚少報告)。 之二、研究PrV是否影響豬嗜中性球對AP的功能反應。 我們發現AP細菌數與細胞數比例為500時引發豬嗜中性球最佳的趨化作用(約30%細胞具趨化性),0.1% Casein也適合作為測試豬嗜中性球的非生物性趨化物。 假性狂犬病病毒能抑制豬嗜中性球非方向性移動,但對於AP趨化性則無顯著影響。 在氧爆作用方面,我們發現 PMA (phorbol 12-myristate 13-diacetate) ,一種Diacylglycerol (DAG) analogue 能引發豬嗜中性球顯著的氧爆作用,以流式細胞分析儀測試其螢光強度達對照組之61倍,病原菌AP、PM同樣能引發氧爆作用4-8倍,且經調理後可達22-34倍。 假性狂犬病病毒能顯著抑制PMA所引發之嗜中性球氧爆作用達50-70%,推論豬假性狂犬病病毒造成臨床上二次性細菌感染症原因之一為抑制嗜中性球氧爆作用產生O2-以及H2O2等殺菌物質的能力。 其機制為抑制DAG (Protein kinase C activator) 活化Protein Kinase C 的路徑所致。 先前研究指出,PrV在豬嗜中性球中複製與抗原表達相當有限,本研究發現經不同處理之PrV,如部份不活化或完全不活化之病毒顆粒同樣造成顯著抑制結果。 推論可能和病毒與接受器 (receptor) 相互作用有關。 另一方面,我們發現此病毒能增加細菌引發的氧爆作用達12-155%,但有時也出現抑制現象下降約15-45%。 因此,PrV如何干擾氧爆作用的機制仍未明瞭須待進一步研究。

Industrial swine production is accompanied by several important viral diseases, exemplified by pseudorabies, hog cholera and porcine reproductive and respiratory syndrome, which are frequently complicated with increased incidence of bacterial superinfections by Actinobacillus pleuropneumoniae (AP), Pasteurella multocida (PM), and Salmonella choleraesuis. One of the hypotheses to explain this clinical finding is that viruses induce polymorphonuclear cells (PMNs, primarily neutrophils) dysfunctions resulted in defective antibacterial resistance. The natural defense functions of neutrophils include chemotaxis, phagocytosis, oxidative burst, and bactericidal activity. Recent evidences accumulated in human pediatrics also suggest the importance of these PMNs functions in specific type of viral infections associated with bacterial complications. The purpose of current study was to use pseudorabies virus (PrV) as a model to explore the possibility of virus-induced PMNs dysfunctions in pigs. The goals of this thesis were: (1) to develop assays for evaluating chemotaxis and respiratory burst of porcine neutrophis, since these assays were still at its infant stage for porcine; and (2) to evaluate whether PrV could affect porcine neutrophil response to AP in ex vivo settings. We found that casein at 0.1% and bacteria AP at sublethal dose served as good chemoattractants. About 30% of porcine neutrophils were able to launch significant chemotaxis to AP, and this response was not affected by PrV. However, PrV did significantly inhibit random migration of porcine neutrophils. We also found that porcine PMNs were able to launch significant (61-fold) oxidative burst to phorbol 12-myristate 13-diacetate (PMA), and to sublethal doses of normal pig serum-opsonized and non-opsonized AP and PM. The PMA-induced burst was inhibited by PrV for 50-70%, and nonviable PrV was as efficient as viable PrV to exert this inhibitory effect. On the other hand, PrV exerted mostly enhancing (from 12 to 155%) and occasionally inhibitory (from 15 to 45%) effects on bacteria-induced oxidative burst. Previous studies indicate that PrV replication and antigen expression are limited in porcine neutrophils. Our findings point out interesting interactions among porcine neutrophils, virus and bacterial pathogen. Our results also suggest that PrV can induce porcine neutrophil dysfunction in a currently unknown mechanism, and further studies are needed to elucidate this mechanism.

List of Figures……………………………………………………VIII
List of Tables……………………………………………………X
Chapter
I.INTRODUCTION……………………………1
II.LITERATURE REVIEW……………………4
1. Neutrophil functions
A. Chemotaxis
Adhesion
Migration
Chemotaxis receptor
Signaling in chemotaxis
Related research in domestic animals
B. Phagocytosis
Receptors involved in phagocytosis
The neutrophil Fc receptors
Fc receptor signaling
Complement receptor 3
Signaling in phagocytosis
Related research in domestic animals
C. Respiratory burst
Signals activating NADPH oxidase
Related research in domestic animals
D. Bactericidal activity
Oxygen-dependent events
Oxygen-independent events
2. Actinobacillus pleuropneumoniae and its Pathogenicity
3. Pasteurella multocida and its pathogenicity
4. Pseudorabies virus and its pathogenicity
5. Virus-induced neutrophil dysfunctions and Virus-bacteria synergism
III.MATERIALS AND METHODS……………53
IV.RESULTS……………………………………61
V.DISCUSSION AND CONCLUSIONS………72
Figures………………………………………………………77
Tables………………………………………………………98
References…………………………………………108

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