鄭謙仁臺灣大學:獸醫學研究所徐晴嵐Hsu, Ching-LanChing-LanHsu2010-05-042018-07-092010-05-042018-07-092009U0001-2707200923110000http://ntur.lib.ntu.edu.tw//handle/246246/178955豬第二型環狀病毒（porcine circovirus type 2; PCV2），為全球豬隻新興疾 -離乳後多系統消耗性症候群（postweaning multisystemic wasting syndrome; MWS）的主要病原。本病為多因子影響的疾病，而血清中含高量 PCV2 核酸 3.9×105 至 1.8×1012 copies/mL) 與臨床發生 PMWS 的嚴重程度有顯著相關性。 年報告指出 Con A 具有促進淋巴球內 PCV2複製且增加淋巴組織病毒力價的現 ，而 PCV2 於血清或淋巴組織中的含量或力價與臨床症狀顯著相關。因此本實 的目的以 Con A 模擬免疫刺激探討豬第二型環狀病毒如何影響刺激下的細胞增 反應。實驗設計包括：一、以自然感染帶原豬，分析不同 PCV2 的病毒含量， Con A 刺激 PBMC 增殖反應之相關性。二、於實驗感染 SPF 豬，探討含高病 豬群的 PBMC 及脾臟細胞，PCV2 superinfected 是否影響 Con A 刺激細胞增 。實驗中細胞增殖反應以流式細胞儀檢測，IFN-γ 及 IL-10 以 ELISA 檢測，病 量以 TagMan® Real-time PCR 測定。細胞的處理分四組包括不處理組，PCV2 ，Con A 刺激組及 Con A/PCV2 處理組。實驗第一部分結果僅 PCV2 uperinfected Con A 刺激之細胞增殖反應與血清及病毒量呈負相關，單獨以 CV2 superinfected 或 Con A 刺激 PBMC 均無顯著差異，而 IL-10 濃度與病毒量 正相關。實驗第二部分結果，於周邊血液細胞，控制組 PCV2 superinfection 有 進 Con A 刺激 PBMC 之細胞增殖反應，但相同處理在攻毒組有抑制細胞增殖 現象，此細胞增殖的抑制與細胞內含高病毒量有關但與細胞之凋亡及壞死無 ，此外也可能與培養上清液 IL-10 濃度有關。在脾臟細胞，控制組中細胞增殖 應、病毒含量及 IL-10 分泌無顯著變化，而 PCV2 superinfection 可促進 Con A 激脾臟分泌 IFN-γ。本研究以 Con A 刺激模擬免疫活化模式，觀察高病毒量或 PCV2 superinfection 可能藉由 IL-10 分泌影響免疫細胞增殖。推論感染豬若經 免疫刺激，則增加病毒複製及 IL-10 分泌，可能使免疫細胞功能受損，而易有 床症狀的產生。Porcine circovirus type 2 (PCV2) is associated with a new emerging disease, ostweaning multisystemic wasting syndrome (PMWS) which causes severe economic osses in swine industry worldwild. PMWS-affected pig is characterized by high PCV2 oad in serum, ranging from 3.9×105 to 1.8×1012 PCV2 copies/mL, which has ignificantly correlated with severe clinical signs of PMWS. Several reports reveal mmune stimulation is an important factor in the induction of PMWS. Con A enhances CV2 replication and high viral titer in PBMC and in lymphoid tissues, respectively, hat has correlated with PMWS as well. The aim of this study is to investigate the mpact of PCV2 infection on Con A stimulated cell proliferative response. The xperimental designs include: 1. To evaluate the correlation between viral load in serum nd Con A stimulated cell proliferation as well as IFN-γ and IL-10 secretions in PBMC rom PCV2-carrier pigs. 2. To evaluate whether PCV2 superinfection affect Con A timulated cell proliferative response in experimental PCV2 infected SPF pigs. Flow ytometry was used to detect cell proliferation response and ELISA was used to valuate the level of IL-10 and IFN-γ. Viral load was measured by TagMan® Real-time CR. Culture of PBMC or spleen cells were separated into 4 group of treatment as ock, PCV2 superinfection, Con A treated and Con A/PCV2. In the first part of the tudy, the tendency of inverse correlation between seral PCV2 load and proliferation esponse of PBMC at Con A/PCV2 group was observed. The postitive correlation etween IL-10 level and viral load was also noted. In the second part of the study, BMC proliferation after Con A stimulation could be enhanced by PCV2 superinfection n the control group. However, this enhancement was not observed in the experimental CV2 infected SPF pigs. In this group, the inhibition of enhanced cell proliferation was orrelated to the cellular viral load but was not associated with the process of necrosis r apoptosis and might be associated with high IL-10 level. In spleen, viral load in pleen cells and IL-10 secretions were insignificant but the level of IFN-γ was enhanced t Con A/ PCV2 group. Our data suggested that high viral load could inibit Con A timulated cell proliferation and the inhibition maybe associated with induction of IL-10.中文摘要 Ⅰ bstract Ⅱ 錄 III 次 V 次 Ⅵ 一章 緒言....................................................................................................................1 二章 文獻回顧.............................................................................................................3 一節 豬環狀病毒.................................................................................................3 -1 環狀病毒的病毒學分類............................................................................3 -2 豬環狀病毒的結構與特性........................................................................3 -3 致裂原 Concanavalin A 影響豬環狀病毒的複製....................................4 -4 豬環狀病毒之感染與組織細胞之分布....................................................5 二節 豬隻離乳後多系統消耗症候群（post-weaning multisystemic）............6 -1 歷史背景與流行病學................................................................................6 -2 臨床症狀與病理變化................................................................................7 -3 PMWS 病程發展.......................................................................................7 -4 PMWS 之致病因子.................................................................................10 三節 細胞免疫反應對抗 PCV2................................................................11 -1 細胞免疫反應..........................................................................................11 -2 細胞激素調節免疫反應..........................................................................13 三章 材料與方法.......................................................................................................18 一節 實驗設計...................................................................................................18 二節 實驗材料...................................................................................................21 -1實驗動物..................................................................................................21 -2 製備豬第二型環狀病毒..........................................................................21 -3 細胞培養液.............................................................................................22 三節 實驗方法...................................................................................................23 -1 豬隻血液單核細胞之分離純化與培養..................................................23 -2 及時定量聚合鏈鎖反應 （Quantitive real-time polymerase chain）...24 -3 檢測病毒特異性的增殖反應..................................................................24 -4 細胞存活率檢測 (Annexin V/ Propidium iodide Apoptotic kit)............26 -5 細胞素的檢測..........................................................................................26 -6 統計分析..................................................................................................26 章 結果.......................................................................................................................28 一節 自然感染帶原PCV2豬之細胞免疫反應與病毒量的相關性...........................................................28 -1.1 PBMC 增殖反應與血液中病毒量的相關性：...................................28 -1.2 IL-10 濃度與血液中病毒量及免疫反應的相關性.............................28 -1.3 PBMC 上清液 IFN-γ 濃度與血液中病毒量的相關性......................29二節 攻毒 SPF 豬 PBMC 免疫反應與細胞內 PCV2 病毒量之關連..............29 -2.1攻毒豬隻血液病毒量及 IL-10 表現....................................................29 -2.2PBMC 之增殖反應................................................................................30 -2.3 PBMC 細胞內病毒量...........................................................................31 -2.4 PBMC 細胞存活率與凋亡率...............................................................32 -2.5 PBMC 之細胞激素分泌.......................................................................32 -2.6細胞病毒量及 IL-10 與 Con A/PCV2 組之增殖反應之關聯..........33 三節 攻毒 SPF 豬脾臟細胞免疫反應與細胞內 PCV2 病毒量之關連...........33 -3.1 脾臟細胞增殖反應...............................................................................35 -3.2 脾臟細胞病毒量...................................................................................35 -3.3 脾臟細胞之細胞激素分泌...................................................................35 五章 討論...................................................................................................................44 六章 文獻回顧...........................................................................................................57application/pdf1013342 bytesapplication/pdfen-USPCV2Concanavalin A白血球間質素-10周邊血液單核細胞淋巴球增殖反應IL-10PBMClymphocyte proliferative responsethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178955/1/ntu-98-R96629012-1.pdf