鄭謙仁Jean, Chian-Ren臺灣大學:獸醫學研究所劉俊廷Liu, Juan-TingJuan-TingLiu2010-05-042018-07-092010-05-042018-07-092008U0001-2407200808133100http://ntur.lib.ntu.edu.tw//handle/246246/178910豬第二型環狀病毒（Porcine circovirus type 2；PCV2），被證實為豬離乳後多系統消耗性症候群（Postweaning multisystemic wasting syndrome；PMWS）的主要病原。其主要病變為廣泛的淋巴腺病、不同程度的淋巴球流失及組織球與多核巨細胞的浸潤。目前PCV2的感染、複製標的細胞及確切致病機制並不清楚，但是活體內研究顯示包含豬肺泡巨噬細胞（Porcine alveolar macrophage；PAM）在內的單核球/巨噬細胞系列是PCV2感染的主要標的細胞，然而體外實驗卻顯示PCV2抗原只存在於PAM的細胞質裡。缺乏存在細胞核內的PCV2抗原訊號，顯示缺乏病毒複製跡象，此結果與野外感染豬隻的感染細胞細胞質及細胞核內病毒抗原高出現率有所牴觸。因此推測應有其他因子的存在，以幫助PCV2在此系列細胞進行複製。脂多醣（lipopolysaccharide；LPS）為革蘭氏陰性菌細胞壁的成分之一，且各菌種之脂多醣體結構各有差異。本實驗室先前證實以E. coli之LPS刺激豬肺泡巨噬細胞（Porcine alveolar macrophage; PAM）可提高其細胞核內PCV2抗原陽性率，並增加其病毒產量。本實驗進ㄧ步嘗試以包括Escherichia coli、Salmonella enterica、Pseudomonas aeruginosa及Klebsiella pneumoniae等菌種的LPS刺激感染PCV2的豬肺泡巨噬細胞，以免疫螢光染色法觀察PCV2抗原分布，結果發現除了E. coli與Salmonella enterica之LPS可誘導PCV2進入PAM細胞核內複製外，對豬非絕對病原菌的Pseudomonas aeruginosa與Klebsiella pneumoniae之LPS亦具有同樣的效果。其細胞培養上清液以同步定量聚合酶連鎖反應（Quantitative real-time PCR reaction）測定，顯示無明顯複製結果之差異；以病毒力價測定顯示PCV2有稍許增加複製之情形。由目前的實驗結果，可見不同LPS皆有些許促使PCV2於PAM內複製之能力，但卻沒有一致的表現。此結果或許可以對PMWS致病機制提供部分的解釋。It has now been demonstrated that porcine circovirus type 2（PCV2） is the causative agent of postweaning multisystemic wasting syndrome（PMWS ). Pigs with PMWS show variable degree of lymphoid depletion with histiocytic and multinucleated giant cell infiltration in the lymphoid organs. Several questions remain unknown including the primary target cells and the permissive cells for the replication of PCV2, as well as the pathogenesis of PMWS in PCV2 infection. In in vivo experiments, monocyte/macrophage lineage cells including porcine alveolar macrophage（PAM）are the major target cells of PCV2. However, there was only high incidence of intracytoplasmic PCV2 signal but lack of intranuclear signals as well as no evidence of PCV2 replication in those cells in vitro. This finding is contradictory to what was found in the tissues of field pigs with PMWS in which both intracytoplasmic and intranuclear PCV2 signals were easily detected in the monocyte/macrophage lineage cells. Concurrent infection and activation of immune system, therefore, have been suggested that may promote PCV2 replication in vivo. Lipopolysaccharide（LPS） is a structural component of the outer membrane of Gram negative bacteria and the composition diversity of LPS between different bacterial species and strains are also observed. It has been demonstrated that LPS of Escherichia coli would induce replication of PCV2 in PAM. In the present study, LPS from different bacterial species, including E. coli, Salmonella enterica, Pseudomonas aeruginosa and Klebsiella pneumoniae, were used as stimulator for PCV2 replication in PAMs and the change in antigen distribution were further evaluated. The results of immunofluorecent assay (IFA) revealed not only the higher virulent LPS of E. coli and Salmonella enterica, but also the LPS from the lower virulent bacterium of non-obligate swine pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae could slightly induce the replication of PCV2 in the nucleus of PAM. The quantitative real-time PCR of the culture supernatant revealed no difference between various treatments. The titration of the culture supernatant revealed mild increase of PCV2 replication in all LPS tested. The results of this study indicate that LPS from different bacterium process varied capability in inducing PCV2 replication in PAM. Nevertheless the level of induction is very mild. The result may provide part of explanation regarding of pathogenesis of PMWS.中文摘要 I文摘要 II錄 IV次 VI次 VII寫對照表 VIII一章 序言 1二章 文獻回顧 3第一節 環狀病毒 3-1 環狀病毒的病毒學分類 3-2 豬環狀病毒結構與特性 3-3 豬環狀病毒的感染以及複製模式 4第二節 離乳後多系統性消耗症 5 2-1 歷史背景與流行病學 5 2-2 臨床症狀與病理變化 6 2-3 PCV2的組織親和性與主要感染的標的細胞 7 2-4 PCV2與免疫的關係 9 2-5 PCV2與其他病原的關係 11 2-6 PCV2相關疾病及症候群 12第三節 細菌脂多醣體（LPS）於病毒感染中所扮演之角色 13 3-1 LPS的特性 13 3-2 LPS刺激巨噬細胞的傳訊路徑 14 3-3 病毒感染與LPS之相互關係 15三章 材料與方法 17第一節 實驗設計 17 1-1 實驗設計流程圖 17二節 實驗材料 19 2-1豬第二型環狀病毒（PCV2）增殖保存 19 2-2 肺泡巨噬細胞採集與培養 19 2-2.1 細胞灌洗液（D-PBS-W） 19 2-2.2 細胞清洗液（RPMI-W） 20 2-2.3 細胞培養液（RPMI-C） 20 2-2.4 肺泡巨噬細胞（Porcine alveolar macrophage; PAM）的採集 20 2-2.5 細菌脂多醣體 21 2-3 豬腎細胞株之培養與病毒力價測試 21 2-3.1 豬腎細胞株（PK-15） 21 2-3.2 PK細胞株培養液（DMEM-C） 22 2-3.3 PK細胞株清洗液（D-PBS） 22 2-3.4 D（+）Glucosamine, Hydrochloride 23 2-3.5 Hank’s Balanced Salt Solutions 23第三節 實驗方法 23 3-1 細胞表面抗原螢光染色法 23 3-2 病毒的感作及LPS的處理 24 3-2.1 豬肺泡巨噬細胞的解凍 24 3-2.2 豬第二型環狀病毒感作 24 3-2.3 不同菌種與濃度之LPS的處理 24 3-2.4 豬肺泡巨噬細胞之細胞質與細胞核病毒陽性率之測定 25 3-3 同步定量聚合酶連鎖反應（Quantitative real-time PCR reaction） 26 3-3.1 上清液收集與DNA之萃取 26 3-3.2 反應溶液成分與條件 26 3-4 病毒力價測定 27 3-5 統計分析 27四章 結果 28第一節 豬肺泡巨噬細胞感染PCV2 28 1-1 豬肺臟沖洗細胞表面抗原染色 28 1-2 豬肺泡巨噬細胞感染PCV2之結果 28第二節 以不同菌種不同濃度之LPS處理豬肺泡巨噬細胞 32 2-1 細胞質內PCV2抗原陽性率之變化 32 2-2 細胞核內PCV2抗原陽性率之變化 32第三節 以不同LPS處理感染PCV2之豬肺泡巨噬細胞 39 3-1 細胞質內PCV2抗原陽性率測定 39 3-2 細胞核內PCV2抗原陽性率測定 39 3-3 上清液中PCV2核酸量測定 40 3-4 上清液中PCV2病毒力價測定 41五章 討論 46六章 參考文獻 56application/pdf1043064 bytesapplication/pdfen-US豬第二型環狀病毒豬肺泡巨噬細胞細菌脂多醣體PCV2porcine circovirus type 2PAMporcine alveolar macrophageLPSlipopolysaccharidethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178910/1/ntu-97-R95629009-1.pdf