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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 龐飛(Victor Fei Pang) | |
dc.contributor.author | Chun-Ming Lin | en |
dc.contributor.author | 林俊明 | zh_TW |
dc.date.accessioned | 2021-06-17T00:14:44Z | - |
dc.date.available | 2017-07-27 | |
dc.date.copyright | 2012-07-27 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-07-05 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65893 | - |
dc.description.abstract | 豬隻第二型環狀病毒(porcine circovirus type 2, PCV2)是一小型、無封套、單股、環狀DNA病毒,其感染廣泛地分布於世界各地。實驗室內以及田間的研究證據指出,PCV2是造成離乳後多系統消耗性症候群(postweaning multisystemic wasting syndrome, PMWS),一種對養豬產業有重大影響的疾病的主要病原;然而,PMWS的發生仍有賴PCV2以外的因子參與。PMWS的特徵為大量的PCV2抗原以及核酸存在於淋巴系統內,伴隨著不等程度的淋巴細胞流失以及肉芽腫性炎症反應。至今,對於PCV2於免疫細胞內複製的確切機制以及PMWS的致病機轉仍不明確。因此,本研究建立一系列活體外(第二、三章)以及活體內(第四、五、六章)模式以嘗試釐清以上議題。
本研究第一部分首先假設免疫活化可誘導PCV2於活化的淋巴細胞內複製,因此利用cocanavalin A (Con A)刺激周邊血液淋巴細胞(peripheral blood lymphocytes, PBLs)作為一活體外模式,以釐清豬隻淋巴細胞對於PCV2的感受性(第二章);其次,藉由在此模式中添加單核球衍伸之樹突細胞(monocyte-derived dendritic cells, MoDCs)以及重組細胞激素,包括介白素(Interleukin)-2、IL-4以及干擾素(interferon-γ),以探討淋巴細胞活化與PCV2複製之間的關聯性(第三章)。結果顯示,在經過致裂原刺激、PCV2感染的PBLs中,細胞內PCV2抗原�核酸帶原率、PCV2核酸量以及感染力價均呈上升的現象,顯示PCV2可感染並於活化的淋巴細胞內複製。藉由表面抗原鑑定顯示:單核球、T以及B細胞都對PCV2的具有感受性,其中又以帶有IgM的B細胞具有相對較高的PCV2陽性率(第二章)。相對於淋巴細胞為主要支持PCV2複製的細胞,MoDCs雖帶有明顯的PCV2抗原但並不顯著地支持PCV2的複製。進一步的研究顯示,IL-2以及樹突細胞的抗原呈現功能是淋巴細胞增生以及PCV2在受感染的淋巴細胞內複製的重要因子(第三章)。 本研究的第二部分探討田間的豬隻在不同程度的PCV2感染下其相關的淋巴結病理變化(第四、五章),並且進而推測其可能的機制(第五、六章)。研究推測,目前例行的原位雜交(in situ hybridization, ISH)以及�或免疫組織化學(immunohistochemical, IHC)染色技術,因受限於本身的靈敏度,可能不足以偵測少量存在於部分免疫細胞的病毒。因此,本研究利用將聚合酶鍊鎖反應(polymerase chain reaction, PCR)以及ISH的步驟結合,建立起更加敏感的間接原位聚合酶鍊反應法(indirect in situ PCR, indirect ISPCR)。藉由ISPCR的建立與使用,可以更有效地偵測於組織內、尤其位於淋巴濾泡的生發中心(germinal center)內的PCV2核酸分布。此外,針對淋巴濾泡中PCV2的感染情形,建立了一套更詳細的分級系統(第四章),以做為後續研究中樣本選擇的標準(第五、六章)。 本研究使用高通量組織微陣列(tissue microarray, TMA),結合半定量ISH/IHC染色以及統計分析模式,以更有效率地分析組織病理變化(第五章)。當PCV2、豬呼吸及生殖症候群病毒以及豬小病毒感染同時存在下,複回歸分析結果支持 不同病毒其各自的致病機制影響PMWS病變的產生,其中以PCV2的效應最為顯著。在PCV2感染所伴隨的淋巴結病變中,B細胞的流失以及巨噬細胞的增殖與浸潤是兩項主要的特徵。除了血循來源以外,淋巴結內T細胞以及巨噬細胞的增殖對於肉芽腫炎症反應的形成佔有重要的地位(第五章)。此外,選擇了7個來自健康、無臨床症狀、帶有PCV2感染的豬隻以及35個來自PMWS患病豬隻的淋巴結,藉由整合多次定量反轉錄聚合酶鏈反應(quantitative reverse transcription PCR, q-rt-PCR)的結果,偵測92個特定免疫基因的表現(第六章)。階層式群集分析法結果顯示,在受到PCV2感染的淋巴結中基因表現呈相大致與不同免疫細胞族群所屬的功能相符合。PCV2感染所伴隨的病變,可歸因於免疫活化的失調以及Th1/Th2的失衡。以主成分分析處理92個選定的基因在42個淋巴結內的表現,顯示52.23%的數據變異性可以被前三項主成分所解釋,顯示PCV2感染所造成的疾病應源自少數主要的因子,但亦受許多次要的因子影響(第六章)。 本研究的證據支持淋巴細胞對於PCV2確實具有感受性(第二、三、四章),根據本研究以及前人的發現,PMWS可能的致病機制為:樹突細胞以及細胞激素所調控的免疫活化可誘發於淋巴細胞內PCV2的複製(第三章),而PCV2對於免疫調節的干擾有利於造成其他病原的共同感染。由於持續或重複抗原的刺激,免疫抑制以及活化之間平衡的失調更趨顯著(第六章),此時,隨著PMWS病程的演進,於淋巴結內呈現高量的PCV2伴隨著淋巴細胞流失以及肉芽腫性炎症反應(第五章)。 | zh_TW |
dc.description.abstract | Porcine circovirus type 2 (PCV2) is a small, non-enveloped, single-stranded, circular DNA virus, and the infection of PCV2 is distributed in swine population worldwide. Experimental and field studies indicate that PCV2 is a required but insufficient causative agent of postweaning multisystemic wasting syndrome (PMWS), a disease that has a significant impact on swine production. The characteristic feature of PMWS is the presence of high levels of PCV2 antigens and nucleic acid in the lymphoid system along with variable lymphoid depletion and granulomatous inflammation. Until now, the precise mechanism regarding the replication of PCV2 in immune cells and the pathogenesis of PMWS are remained to be elucidated. Therefore, serial in vitro (Chapters II and III) and in vivo (Chapters IV, V and VI) models were performed in the present study to clarify above issues.
In the first portion of the present study, it is hypothesized that immune activation induces PCV2 replication in activated lymphocytes. Therefore, concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) was used as an in vitro model to clarify the susceptibility of swine lymphocytes to PCV2 (Chapter II). Subsequently, this model was further expanded by the addition of monocyte-derived dendritic cells (MoDCs) and recombinant cytokines, including interleukin 2 (IL-2), IL-4, and interferon γ, to determine the correlations between lymphocyte activation and PCV2 replication (Chapter III). The evidences of increase in cellular PCV2 antigen- and/or nucleic acid-containing rates, PCV2 genome copy number, and titer of infectious PCV2 in mitogen-stimulated, PCV2-infected PBLs support that PCV2 infects and replicates in activated swine lymphocytes. By phenotyping the PCV2 antigen-positive cells, it was revealed that T and B lymphocytes as well as monocytes were susceptible to PCV2 infection; however, IgM-positive B lymphocytes appeared to have a relatively higher PCV2-positive rate (Chapter II). While lymphocytic cells served as a major site of PCV2 replication, MoDCs harbored a significant amount of PCV2 antigens but did not actively sustain the replication of PCV2. Further study has demonstrated that IL-2 and the accessory cell function of DCs were key factors required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes (Chapter III). In the second portion of the present study, serials in vivo studies were conducted to characterize the changes in inguinal LNs in the naturally PCV2-infected pigs (Chapters IV and V) and to infer the possible mechanism of PCV2-associated lymphoid lesion developed (Chapter V and VI). It is speculated that limited by their sensitivity, conventional in situ hybridization (ISH) and/or immunohistochemical (IHC) staining may not be sufficient to detect few copies of PCV2 contained in a portion of immune cells. Therefore, a more sensitive method, indirect in situ polymerase chain reaction PCR (ISPCR), was developed through the combination of PCR and ISH procedure (Chapter IV). Using the indirect ISPCR, signals of PCV2 nucleic acid, especially those in germinal centers, could be more effectively detected. In addition, a detailed grading system was proposed to categorize the pattern of natural PCV2 infection in lymphoid follicles (Chapter IV) and used as criteria for sample selection in the following studies (Chapters V and IV). To obtain tissue-based information effectively, a new model of high-throughput tissue microarray (TMA) in conjunction with semi-quantified ISH/IHC staining and statistic analysis was established (Chapter V). The results of multiple regression analysis showed that each of PCV2, porcine respiratory and reproductive syndrome virus (PRRSV), and porcine parvovirus (PPV) had its own contributions on the development of lymphoid lesions in PMWS with PCV2 as the major causative agent. B lymphocyte depletion and macrophage proliferation and infiltration are the two hallmarks of PCV2-associated lymphoid lesions. Apart from blood recruitment, local T cell and macrophage proliferation may play a crucial role on the development of granulomatous inflammation (Chapter V). Furthermore, the expressions of 92 selected immune genes in 7 and 35 inguinal LNs obtained from healthy subclinically PCV2-infected and PMWS-affected pigs were assessed by the integration of several quantitative reverse transcription polymerase chain reaction experiments (Chapter IV). Using hierarchical cluster analysis, the gene expression profiles in these PCV2-infected LNs were generally compatible with the divergent functions of different immune cell populations. Aberrant immune activation and imbalanced Th1/Th2 orientation are considered to contribute to the development of PCV2 infection-associated lymphoid lesions. Principle component analysis of the expression profile of the 92 selected immune genes in the 42 above mentioned LNs revealed that 52.23% of the total data variants could be explained by the top-3 principle components, suggesting that the disease development of PCV2 infection may be associated with a few major and some minor factors (Chapter VI). In conclusion, evidences from the present study suggest that lymphocytes are indeed susceptible to PCV2 (Chapters II, III and IV). Immune activation modulated by DCs and cytokines triggers the replication of PCV2 in lymphocytes (Chapter III). Subsequently, the perturbation of immune regulation by PCV2 facilitates the co-infection of other pathogens. Due to the persistent or repeated episodes of antigenic stimulation, the balance between immune suppression and activation is further perturbed (Chapter VI). As the progression of PMWS development, lymphoid depletion and granulomatous inflammation occur in the LNs bearing heavy PCV2 load (Chapter V). | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T00:14:44Z (GMT). No. of bitstreams: 1 ntu-101-D95629003-1.pdf: 6066273 bytes, checksum: 6c578fc3c4edb4b1728d4505e25031fb (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 目錄 Contents
中文摘要…………………………………………………………I Abstract………………………………………………………IV Chapter I General introduction……………………………1 Chapter II Characterization of porcine circovirus type 2 (PCV2) infection in swine lymphocytes using mitogen-stimulated peripheral blood lymphocytes from healthy PCV2-carrier pigs Vet. Immunol. Immunopathol., 2008, 124, 355–366 …………21 Chapter III Monocyte-derived dendritic cells enhance cell proliferation and porcine circovirus type 2 replication in concanavalin A-stimulated swine peripheral blood lymphocytes in vitro Vet. Immunol. Immunopathol., 2012, 45, 368–378………34 Chapter IV Development and evaluation of an indirect in situ polymerase chain reaction for the detection of porcine circovirus type 2 in formalin-fixed and paraffin-embedded tissue specimens Vet. Microbiol., 2009, 138, 225–234………46 Chapter V Immunopathological characterization of porcine circovirus type 2 infection-associated follicular changes in inguinal lymph nodes using high-throughput tissue microarray Vet. Microbiol., 2011, 149, 72–84 …………57 Chapter VI Immune gene expression profiles in swine inguinal lymph nodes with different viral loads of porcine circovirus type 2 Manuscript in preparation…71 Chapter VII General discussion……108 | |
dc.language.iso | en | |
dc.title | 使用活體內以及活體外模式探討豬第二型環狀病毒於免疫細胞以及淋巴組織的感染 | zh_TW |
dc.title | Characterization of porcine circovirus type 2 infection in immune cells and lymphoid organs by using in vitro and in vivo models | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 鄭謙仁(Chian-Ren Jeng) | |
dc.contributor.oralexamcommittee | 李維誠,張志成,劉涓,邱明堂 | |
dc.subject.keyword | 豬第二型環狀病毒,淋巴細胞,免疫, | zh_TW |
dc.subject.keyword | porcine circovirus type 2,lymphocyte,immune, | en |
dc.relation.page | 128 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-07-06 | |
dc.contributor.author-college | 獸醫專業學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
顯示於系所單位: | 獸醫學系 |
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ntu-101-1.pdf 目前未授權公開取用 | 5.92 MB | Adobe PDF |
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