臺灣博碩士論文加值系統

豬生殖與呼吸綜合症病毒 ( porcine reproductive and respiratory syndrome virus; PRRSV ) 造成台灣豬場極大經濟損失，目前仍無有效的控制方法。樹狀突細胞 ( Dendritic cells ; DCs ) 由骨髓造血幹細胞分化而來，在免疫反應之起始及維持上扮演著關鍵性的角色。本研究將 PRRSV 感染未成熟樹狀突細胞 ( imDCs )，利用流式細胞儀檢測其感染率、表面抗原、細胞凋亡、吞噬作用以及利用酵素結合免疫吸附分析 ( Enzyme-Linked Immunosorbent Assay；ELISA ) 檢測imDCs受PRRSV 感染後細胞激素的產生情形。結果顯示，PRRSV可以感染imDCs並且進行複製。PRRSV感染imDCs之後細胞之突起增加，細胞核呈現核染質著邊現象，細胞凋亡的比例也顯著 ( P<0.05 ) 增加。PRRSV感染imDCs之後，CD80/86的表現顯著 ( P<0.05 ) 上升，而MHCⅠ的表現顯著 ( P<0.05 )下降。PRRSV感染imDCs之後，可誘導IL-10的產生，而抑制細胞對不可溶性抗原的攝入能力。

Porcine reproductive and respiratory syndrome virus ( PRRSV ) has invaded the pig herds in Taiwan and causes remarkable economic losses to the pig farmers. Currently, no effective method is available for the control of this disease. Dendritic cells originate from bone marrow stem cells, play crucial roles in activation of naïve and resting antigen-specific T cells. In this study, the infectivity of PRRSV in immature dendritic cells ( imDCs ), phenotypic and functional alterations including surface antigen expression , apoptosis and phagocytosis ability of imDCs after the infection of PRRSV were monitored by flow cytometry. ELISA was used to detect cytokine secretion in imDCs after the infection of PRRSV. The infection of PRRSV in imDCs resulted in a productive replication in the cells. The infected cells had increased cell process and displayed condensation of chromatin, increased expression of CD80/86 but decreased expression of MHCⅠ. Infection of imDC with PRRSV induced IL-10 production but down-regulated its ability to uptake the insoluble antigen.

目錄
中文摘要........................................................................................................ Ⅰ
Abstract.......................................................................................................... Ⅱ
誌謝………………………………………………………………………. Ⅳ
目錄............................................................................................................. Ⅴ
圖表目錄..................................................................................................... Ⅶ
第1章 前言.................................................................................................... 1
第2章 文獻回顧............................................................................................ 3
2. 1 豬生殖與呼吸道綜合症病毒.............................................................. 3
2. 2 PRRSV致病機制…………………………….................................... 5
2. 2. 1 PRRSV進入易感受性細胞之方式…......................................... 5
2. 2. 2 PRRS的致病機制……………………….................................... 5
2. 2. 3 PRRSV對於生殖的影響………………………………………. 6
2. 2. 4 PRRSV對細胞凋亡的影響…………….................................... 6
2. 3 PRRSV感染後對免疫反應的影響…................................................. 7
2. 3. 1 先天性免疫反應…….................................................................. 7
2. 3. 2 體液性免疫反應…….................................................................. 8
2. 3. 3 細胞性免疫反應……….............................................................. 8
2. 4 豬隻樹狀突細胞…………………………………............................... 9
2. 4. 1 樹狀突細胞的生命史.................................................................. 9
2. 4. 2 樹狀突細胞的細胞特徵……………………………………….. 10
2. 4. 3 樹狀突細胞之吞噬能力.............................................................. 11
2. 5 樹狀突細胞對病毒的感受性…........................................................... 11
2. 5. 1 人類病毒研究方面….................................................................. 12
2. 5. 2 豬隻病毒研究方面……….......................................................... 13
第3章 材料與方法....................................................................................... 15
3. 1 實驗動物……...…….......................................................................... 15
3. 2 豬隻骨髓造血幹細胞衍生之未成熟樹狀突細胞 ( immature dendritic cells ; imDC ) 之分離、純化及培養…………………..... 15
3. 3 肺泡巨噬細胞…………………………………………………….… 16
3. 4 豬生殖與呼吸綜合症病毒 ( PRRSV ) 來源與病毒力價測定…... 16
3. 5 以PRRSV感染imDC和肺泡巨噬細胞………………….……..... 16
3. 6 PRRSV感染imDC後的病毒檢測…………………………….….. 17
3. 6. 1 以流式細胞儀檢測豬生殖與呼吸綜合症病毒 ( PRRSV )....... 17
3. 6. 2 間接螢光染色……...................................................................... 17
3. 6. 3 電子顯微鏡檢查.......................................................................... 18
3. 7 imDC以PRRSV感染後其細胞大小變化檢測……….………….. 18
3. 8 細胞表面抗原檢測……………………………………………...….. 18
3. 9 以PRRSV感染後之細胞存活與凋亡檢測……………………….. 19
3. 10 吞噬功能檢測................................................................................. 19
3. 10.1 imDC吞噬顆粒性抗原能力之檢測........................................... 19
3. 10.2 imDC攝入及處理可溶性抗原能力之檢測............................... 20
3. 11 細胞素檢測..................................................................................... 20
3. 12 統計分析......................................................................................... 21
第4章 結果................................................................................................... 22
4. 1 豬生殖與呼吸綜合症病毒對imDC和AM的感受性..................... 22
4. 2 PRRSV感染imDC後其形態之變化............................................. 22
4. 3 PRRSV感染imDC後對細胞大小的影響..................................... 27
4. 4 PRRSV感染imDC及AM後對細胞存活的影響......................... 28
4. 5 PRRSV感染對imDC表面抗原之影響......................................... 28
4. 6 PRRSV感染imDC後對細胞分泌細胞激素的影響..................... 32
4. 7 PRRSV感染對imDC吞噬功能之影響......................................... 32
第5章 討論.................................................................................................. 35
參考文獻…………………………………………………………….……... 40
作者簡介………………………………………………………….………... 50

參考文獻
吳家彰。2005。豬樹狀突細胞受假性狂犬病病毒感染後其表現型及功能的變化。國立屏東科技大學獸醫學研究所碩士論文。
張志成、鍾文彬、林敏雯、翁仲男、楊平政、邱雲棕、張文發、朱瑞民。臺灣地區豬繁殖與呼吸道症候群 I. 病毒分離。中華民國獸醫學會雜誌。19:268-276。1993。
莊秀琪、陳昱彰、鍾文彬、吳彰哲。探討豬隻骨髓造血母細胞試管內分化為樹狀突細胞之細胞特徵。台灣獸醫誌。31: 195-207。2005。
Aasted B, Bach P, Nielsen J, Lind P. Cytokine profiles in peripheral blood mononuclear cells and lymph node cells from piglets infection in utero with porcine reproductive and respirtary syndrome virus. Clin Diagn Lab Immunol 9: 1229-1234, 2002.
Abul K, Abbas K, Andrew H. Lichtman K. Cell and Molecular Immunology. Fifth edition. 243-251, 2000.
Albina E, Piriou L, Hutet H. Immune responses in pigs infected with porcine reproductive and respiratory syndrome virus ( PRRSV ). Vet Immunol Immunopathol 61: 49-66, 1998.
Alcami A. Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol 3: 36-50, 2003.
Ardavun C, Martinez de Hoyo G, Martin P, Anjuere F, Arias CF, Marin AR,Ruiz S, Parrillas V, Hernandez H. Origin and differentiation of dendritic cells. Trends Immunol 22: 691-700, 2001.
Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K. Immunobiology of dendritic cells. Annu Rev Immunol 18: 767-811, 2000.

Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 392: 245-52, 1998.
Bautista EM, Ferman GS, Gregg D, Brum MC, Grubman MJ, Golde WT. Constitutive expression of alpha interferon by skin dendritic cells confers resistance to infection by foot-and-mouth disease virus. J Virol 79: 4838-4847, 2005.
Bautista EM, Gregg D, Golde WT. Characterization and functional analysis of skin-derived dendritic cells from swine without a requirement for in vitro propagation. Vet Immunol Immunopathol 88: 131-148, 2002.
Buddaert W, van Reeth K, Pensaert MB. In vivo and in vitro interferon ( IFN ) studies with the porcine reproductive and respiratory syndrome virus ( PRRSV ). Adv Exp Med Biol 440: 461-467, 1998.
Canon N, Audige L, Denac H, Hofmann M, Griot C. Evidence of freedom from porcine reproductive and respiratory syndrome virus infection in Switzerland. Vet Rec 142: 142-143, 1998.
Carrasco CP, Rigden RC, Schaffner R, Gerber H, Neuhaus V, Inumaru S, Takamatsu H, Bertoni G, McCullough KC, Summerfield A. Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties. Immunology 104: 175-184, 2001.
Carrasco CP, Rigden RC, Vincent IE, Balmelli C, Ceppi M, Banhofer O, Tache V, Hjertner B, McNeilly F, Gennip HG, McCullough KC, Summerfied A. Interaction of classical swine fever virus with dendritic cells. J Gen Virol 85: 1633-1641, 2004.
Carter QL, Curiel RE. Interleukin-12 ( IL-12 ) ameliorates the effects of porcine respiratory and reproductive syndrome virus ( PRRSV ) infection. Vet Immunol Immunopathol 107: 105-118, 2005.
Cavanagh, D. Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol 142:629-633, 1997.
Choi C, Chae C. Expression of tumour necrosis factor-αis associated with apoptosis in lungs of pigs experimentally infected with porcine reproductive and respiratory syndrome virus. Res Vet Sci 72: 45-49, 2002.
Delputte PL, Nauwynck HJ. Porcine arterivirus infection of alveolar macrophages is mediated by sialic acid on the virus. J Virol 78: 8094-8101, 2004.
Drake DR, Moser JM, Hadley A, Altman JD, Maliszewski C, Butz E, Lukacher AE. Polyomavirus-infected dendritic cells induce antiviral CD8+ lymphocyte. J Virol 74: 4093-4101, 2000.
Duan X, Nauwynck HJ, Dendaert MB. Effects of origin and susceptibility to porcine reproductive and respiratory syndrome virus ( PRRSV ). Acrh Virol 142: 2483-2497, 1997.
Duan X, Nauwynck HJ, Favoreel HW, Pensaert MB. Identification of a putative receptor for porcine reproductive and respiratory syndrome virus on porcine alveolar macrophages. J Virol 72:4520-4523, 1998.
Fantuzzi L, Purificato C, Donato K, Belardelli F, Gessani S. Human immunodeficiency virus type gp120 induces abnormal maturation and functional alterations of dendritic cells: a novel mechanism for AIDS pathogenesis. J Virol 78: 9763-9772, 2004.
Fonteneau JF, Kavanagh DG, Lirvall M, Sanders C, Cover TL, Bhardwaj N, Larsson M. Characterization of the MHC class I cross-presentation pathway for cell-associated antigens by human dendritic cells. Blood 102: 4448-4455, 2003.
Fugier-Vivier I, Servet-Delprat C, Rivailler P, Rissoan MC, Liu YJ, Rdourdin-Combe C. Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells. J Exp Med 186: 813-823, 1997.
Gagnon CA, Lachapelle G, Langelier Y, Massie B, Dea S. Adenoviral expressed GP5 of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions. Arch Virol 146: 951-972, 2003.
Garner M, Gleeson GLJ, Holyoade PK, Cannon RM, Doughty WJ. A national serological survey to verify Australia’s freedom from procine reproductive and respiratory syndrome. Aust Vet J 75: 596-600, 1997.
Goyal SM. Porcine reproductive and respiratory syndrome. J Vet Diagn Inest 5: 656-664, 1993.
Hengel H, Reusch U, Geginat G, Ho KL, Tappels R, Ruppert T, Hellebrand E, Koszinowski UH. Macrophages escape inhibition of major histocompatibility complex class I-dependent antigen presentation by cytomegalovirus. J Virol 17: 7861-7868, 2000.
Hill H. Overview and history of mystery swine disease ( swine infertility and respiratory syndrome ). Proc Mystery Swine Dis Comm Meet. 29-31, 1990.
Ho LJ, Wang JJ, Shaio MF, Kao CL, Chang DM, Han SW, Lai JH. Infection of human dendritic cells by dengue virus causes cell maturation and cytokine production. J Immunol 166: 1499-1506, 2001.
Hopper SA, White MEC, Twiddy N. An outbreak of blue-eared pig disease ( porcine reproductive and respiratory syndrome ) in four pig herds in Great Britain. Vet Rec 131: 140-144, 1992.
Inaba K, Turley S, Iyoda T. The formation of immunogenic major histocompatibility complex class II–peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. J Exp Med 191: 927-936, 2000.
Kim TS, Benfied DA, Rowland RR. Porcine reproductive and respiratory syndrome virus-induced cell death exhibits features consistent with a nontypical frome of apoptosis. Virus Res 85: 228-232, 2002.
Klagge IM, Schneider-Schaulies S. Virus interation with dendritic cells. J Gen Virol 80: 823-833, 1999.
Kreutz LC, Ackermann MR. Porcine reproductive and respiratory syndrome virus enters cells through a low pH-dependent endocytic pathway. Virus Res 42: 137-147, 1996.
Kreutz LC. Cellular membrane factors are the major determinants of porcine reproductive and respiratory syndrome virus tropism. Virus Res 53: 121-128, 1998.
Labarque G, Van Gucht S, Nauwynok H, Van Reeth K, Pensaert M. Apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines. Vet Res 34: 249-260, 2003.
Labarque GG, Nauwynck HJ, Van Reeth K. Effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs. J Gen Virol 81: 1327-1334, 2002.
Lee C, Hodgin D, Calvert JG, Welch SK, Jolie R, Yoo D. Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication. Virology 346: 238-250, 2006.
Lee SM, Kleiboeker SB. Porcine arterivirus activates the NF-κB pathway through IκB degradation. Virology 132: 45-52, 2005.
Liu A, Takahashi M, Narita M, Zheng Z, Kanazawa N, Abe T, Nikkuni K, Furukawa T, Toba K, Fuse I, Aizawa Y. Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment. J Immunol Methods 261: 49-63, 2002.
Lopez-Fuertes L, Campos E, Domenech N. Porcine reproductive and respiratory syndrome ( PRRSV ) virus down-modulates TNF-alpha production in infected macrophages. Virus Res 69: 41-44, 2000.
Lopez-Fuertes L, Domench N, Alvarez M. Analysis of cellular immune response in pigs recovered from porcine reproductive and respiratory syndrome infection. Virus Res 64: 33-42, 1999.
Lopez OJ, Osorio FA. Role of neutralizing antibodies in PRRSV protective immunity. Vet Immunol Immunopathol 102: 155-163, 2004.
Makala LH, Haverson K, Stokes CR, Bailey M, Bland PW. Isolation and characterization of pig peyer’s patch dendritic cells. Vet Immunol Immunopathol 16: 67-81, 1998.
Mardassi HB, Massie C, Dea S. Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome. Virology 221: 98-112, 1996.
Mardassi HR, Athanassious S, Mounir LH, Dea S. Porcine reproductive and respiratory syndrome: morphological, biochemical and serological characteristics of Quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome. Can J Vet Res 58: 55-64, 1994.
McCullough KC, Basta S, Knotig S, Gerber H, Schaffner R, Kim YB, Saalmuller A, Summerfield A. Intermediate stages in monocyte-macrophage differentiation modulate phenotype and susceptibility to virus infection. Immunology 98: 203-212, 1999.
McCullough KC, Schaffner R, Natale V, Kim YB, Summerfield A. Phenotype of porcine monocytic cells: modulation of surface molecule expression upon monocyte differentiation into macrophages. Vet Immunol Immunopathol 58: 265-275, 1997.
Meulenberg JJ, de Meijer MEJ, Moormann RJM. Subgenomic RNAs of Lelystad virus contain a conserved leader-body junction sequence. J Gen Virol 74: 1697-1701, 1993.
Meulenberg JJ, Petersen den Besten A, de Kluyver E, van Nieuwstadt A, Wensvoort G, Moormann RJ. Molecular characterization of Lelystad virus. Vet Microbiol 55: 197-202, 1997.
Mikloska Z, Bosnjak L, Cunningham AL. Immature monocyte-derived dendritic cells are productively infected with herpes simplex virus type 1. J Virol 75: 5958-5964, 2001.
Miller LC, Fox JM. Apoptosis and porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol 102: 131-142, 2004.
Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19: 683-765, 2001.
Oleksiewicz MB, Nielsen J. Effect of porcine reproductive and respiratory syndrome virus ( PRRSV ) on alveolar lung macrophage survival and function. Vet Microbiol 66: 15-27, 1999.
Paillot R, Laval F, Audonnet JC, Andreoni C, Juillard V. Functional and phenotypic characterization of distinct porcine dendritic cells derived from peripheral blood monocytes. Immunology 102: 396-404, 2001.
Pollara G, Speidel K, Samady L, Rajpopat M, McGrath Y, Ledermann J, Coffin RS, Katz DR, Chain B. Herpes simplex virus infection of dendritic cells: balance among activation, inhibition, and immunity. J Infect Dis 187: 165-178, 2003.
Prieto C, Garcia C, Simarro I, Castro JM. Temporal localization of porcine reproductive and respiratory syndrome virus in reproductive tissues of experimentally infected boars. Theriogenology 60: 1505-1514, 2003.
Prieto CR, Sanchez S, Maritn-Rillo P, Suarez I, Simarro A, Solana CC, Castro JM. Exposure of gilts in early gestation to porcine reproductive and respiratory syndrome virus. Vet Rec 138: 536-539, 1996.
Rossow KD, Collins JE, Goyal SM, Nelson EA, Christopher-Hennings J, Benfield DA. Pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs. Vet Pathol 32: 361-373, 1995.
Salmon H, Johnson I, Germana S, Haller GW, Sachs DH, Leguern C. Dendritic cells enrich from swine thymus co-express CD1, CD2 and major histocompatibility complex class II and actively stimulate alloreactive T lymphocytes. Scand Immunol 52: 164-172, 2000.
Samsom JN, de Bruin TG, Voermans JJ. Changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus : a role for CD8+ cells. J Gen Virol 81: 497-505, 2000.
Siavoshian S, Abraham JD, Thumann C, Kieny MP, Schuster C. Hepatitis C virus core, NS3, NS5A, NS5B proteins induce apoptosis in mature dendritic cells. J Med Virol 75: 402-411, 2005.
Sirinarumitr T, Zhang Y, Kluge JP, Halbur PG, Paul PS. A pneumo-virulent United States isolate of porcine reproductive and respiratory syndrome virus induces apoptosis in bystander cells both in vitro and in vivo. J Gen Virol 79: 2989-2995, 1998.
Snijder EJ, Meulenberg JJ. The molecular biology of arteriviruses. J Gen Virol 79: 961-979, 1998.
Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9: 271-296, 1991.
Suarez PM, Diaz-Guerra C, Prieto M, Esteban JM, Castro A, Nieto P, Ortin J. Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis. J Virol 70: 2876-2882, 1996.
Suradhat S, Thanawangnuwech R, Poovorawan Y. Upregulation of IL-10 gene expression in porcine peripheral blood monuclear cells by porcine reproductive and respiratory syndrome virus. J Gen Virol 84: 453-459, 2003.
Van Alstine WG, Kanitz CL, Stevenson GW. Time and temperature survivability of PRRSV virus in serum and tissues. J Vet Diagn Invest 5: 621-622, 1993.
Van Gucht S, Labarque G, Van Reeth K. The combination of PRRS virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs. Vet Immunol Immunopathol 102: 165-178, 2004.
Van Gucht S, Van Reeth K, Pensaert M. Interation between porcine reproductive and respiratory syndrome virus and bacterial endotoxin in the lungs of pigs: potentiation of cytokine production and respiratory disease. J Clin Microbiol 41: 960-966, 2003.

Vincent IE, Carrasco CP, Hermann B, Mechan BM, Allen GM, Summerfield A, McCullough KC. Dendritic cells harbar infectious porcine circovirus type 2 in the absence of apparent cell modulation or replication of the virus. J Virol 77: 13288-13300, 2003.
Vossen MT, Westerhout EM, Soderberg-Naucler C, Wiertz EJ. Viral immune evasion: a masterpiece of evolution. Immunogenetics 54: 527-542, 2002.
Wensvoort GC, Terpstra JM, Pol A, ter Laak EA, Bloemraad M, de Kluyver EP, Kragtern C, van Buiten L, den Besten A, Wagenaar FJ, Broekhuijden M, Moonen PL, Zetstra T, A de Boer E, Tibben JH, ed Jong MF, van ‘t Veld P, Groenland GJR, van Gennep JA, Voets MT, Verheijden JHM, Braamskamp J. Mystery swine disease in the Netherlands:the isolation of Lelystad virus. Ver Q 13: 121-130, 1991.
Wootton S, Yoo D, Rogan D. Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus ( PRRSV ) isolate. Arch Virol 145: 2297-2323, 2000.
Xiao Z, Batista L, Dee S, Halbur P, Murtaugh MP. The Level of virus-specific T-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load. J Virol 78: 5923-5933, 2004.
Yoon KJ, Wu LL, Zimmerman JJ, Hill HT, Platt KB. Antibody-dependent enhancement ( ADE ) of porcine reproductive and respiratory syndrome virus ( PRRSV ) infection in pigs. Vir Immunol 9: 51-63, 1996.
Yoon KJ, Zimmerman JJ, Swenson SL, McGinley MJ, Eernisse KA, Brevik A, Rhinehart LL, Frey ML, Hill HT, Platt KB. Characterization of the humoral immune response to response to porcine reproductive and respiratory syndrome ( PRRS ) virus infection. J Vet Diagn Invest 7: 305-312, 1995.