鄭謙仁臺灣大學:獸醫學研究所陳亮君Chen, Liang-JiunLiang-JiunChen2007-11-282018-07-092007-11-282018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/59976離乳後多系統消耗性症候群(Postweaning multisystemic wasting syndrome; PMWS)主要發生於5-12週齡的豬隻,其引起的病原為第二型豬環狀病毒(Porcine circovirus type 2;PCV2);PCV2與其他細菌及病毒的混合感染或患畜經過免疫刺激後常會增加此疾病的嚴重程度。目前PCV2確切的致病機制及其感染和複製的標的細胞仍不清楚,雖然在活體內PCV2感染之標的細胞主要為單核/巨噬細胞及抗原呈現細胞,但在活體外的試驗卻無法證實PCV2會在此類細胞進行複製。本研究為瞭解PCV2複製之機制,以豬肺泡巨噬細胞(PAMs)及細菌的脂多醣(LPS)進行PCV2活體外複製之實驗;利用抗PCV2高力價豬血清及抗PCV2 ORF1抗體進行免疫螢光染色(IFA),結果於LPS刺激後之PCV2感染PAMs的細胞核內出現陽性訊號;使用反轉錄聚合酶鏈鎖反應亦可偵測出PCV2 spliced Cap mRNA,其細胞培養上清液也可見病毒力價的上升,但同樣情形並不見於未經LPS處理之PCV2感染之PAMs。本實驗結果顯示經由適當的LPS刺激後,PCV2可在感染PAMs細胞內進行複製。另外,於LPS處理之PCV2感染組的細胞培養上清液,有較多量的IL-1beta、IL-8、IL-10及TNF-alpha等蛋白質表現。雖然LPS如何啟動PCV2複製的機制仍不清楚,但報告指出脂多醣刺激人類免疫缺陷病毒(Human immunodeficiency virus;HIV)感染之單核巨噬細胞,是經由細胞內Nuclear factor-kappaB(NF-κB)的活化,引起不同的功能反應而增加HIV的複製。因此使用PK-15細胞作為一模式,瞭解NF-κB的活化與PCV2複製是否存著關連性。本實驗分別使用NF-κB activation inhibitor及Phorbol 12-myristate 13-acetate(PMA)促使NF-κB抑制及活化,經PMA及NF-κB activation inhibitor各別處理之PK-15細胞皆可輕微增加PCV2病毒力價,而當PMA及NF-κB activation inhibitor共同處理之PK-15細胞則可見明顯增加PCV2病毒力價。本實驗結果說明了革蘭氏陰性菌於PCV2複製中,可能扮演了一個很重要的角色,且亦可改變免疫反應前細胞激素相(proinflammatory cytokines profiles)。於PK-15細胞株細胞模式試驗中得知NF-κB的活化與PCV2複製並不具有直接關聯性,但NF-κB的活化或抑制似乎是會藉由間接作用影響PCV2複製,然其真正的活化機序則有待更進一步的實驗探討。Porcine circovirus type 2(PCV2) is the necessary causative agent of postweaning multisystemic wasting syndrome(PMWS) in swine; however, a variety of co-factors including other infectious agents, are thought to be necessary for the full development of the disease. PCV2 nucleic acid and/or antigens are consistently observed in cells of monocyte/macrophage lineage and antigen-presenting cells in the lesions of PMWS-affected pigs; but no evidence of PCV2 replication was observed in these cells in vitro. To pursuit the question regarding the mechanism of PCV2 replication, porcine alveolar macrophages(PAMs)and lipopolysaccharide(LPS) were used in this study. The results revealed intranuclear signal of PCV2 and ORF1 protein were only detected in E. coli’s polysaccharide(LPS)-treated PCV2-inoculated PAMs. The PCV2 replication products corresponding to PCV2 spliced Cap mRNA and increased levels of infectious virus progeny were also successfully demonstrated in the LPS-treated PCV2-inoculated PAMs, but not in the mock LPS-treated PCV2-inoculated PAMs. The mechanism of LPS triggering PCV2 replication is not understood but may support the hypothesis that LPS is a stimulator of PCV2 replication which may contribute to the full development of PMWS in pigs during appropriate bacteria co-infection. In the HIV studies, LPS stimulation enhances the replication of HIV in monocytic-macrophage linage cells. In this study, IL-1beta、IL-8、IL-10 and TNF-alpha expressions were notably increased in the LPS-treated PCV2-inoculated PAMs when compared to the mock LPS-treated PCV2-inoculated PAMs. The stimulation of LPS is related to the activation of NF-κB which is a transcription factor with diverse functions. In the subsequent study, attempts to induce NF-κB activation and inhibition by PMA and NF-κB activation inhibitor, respectively, in PK-15 cells were proceed to investigate the possible involvement of NF-κB activation in PCV2 replication. The result showed that virus titer was mildly increased in the PMA-treated PCV2-infected PK-15 cells and NF-κB activation inhibitor-treated PCV2-infected PK-15 cells when compared to the mock-treated PCV2-infected PK-15 cells. The virus titer was notably increased in the PMA- and NF-κB activation inhibitor co-treated PCV2-infected PK-15 cells when compared to the PCV2-infected PK-15 cells. The findings suggest that Gram-negative bacteria may be an important factor in PCV2 replication. Additionally, the proinflammatory cytokine profiles are also affected by bacterial lipopolysaccharide. From the results of PK-15 cells, the inhibition of NF-κB activation may indirectly relate to the PCV2 replication.中文摘要 I 英文摘要 III 目錄 V 表次 VIII 圖次 IX 第一章、緒言 1 第二章、文獻回顧 3 第一節 離乳後多系統性消耗性症候群(PMWS) 3 1-1 歷史背景及發生概況 3 1-2 病毒特牲 5 1-3 PMWS引起之臨床症狀和病理變化 7 第二節 PMWS之致病機制 9 2-1 PCV2之細胞親和性及標的細胞分布情形 9 2-2 PCV2的複製機制 11 2-3 PCV2與免疫的關係 13 2-4 PCV2與其他病原之相互關係 15 2-5 PCV2與細胞激素之相關性 18 第三節 細菌粘多醣體(LPS)於病毒感染中所扮演之角色 19 3-1 病毒感染與LPS之相互關係 19 第四節 NF-κB於病毒感染中所扮演之角色 20 4-1 NF-κB之介紹 20 4-2 NF-κB之功用 21 4-3 病毒感染與NF-κB之關係 22 第三章、材料與方法 25 第一節 試管內豬肺泡巨噬細胞試驗 25 (一)實驗設計及流程圖 25 (二)實驗材料 27 1. 第二型豬環狀病毒 (PCV2) 27 2. 細胞培養液 (RPMI-C) 28 3. 細胞清洗液 (RPMI-W) 28 4. 細菌脂多醣 29 5. 豬肺臟巨噬細胞 (Porcine alveolar macrophages;PAMs) 之採集 29 (三)實驗方法 30 一、LPS之處理 30 1. 第二型豬環狀病毒 (PCV2) 感作 30 2. 不同濃度之LPS比較 31 3. 豬肺臟巨噬細胞之核內病毒陽性率之測定 31 二、PCV2的複製 32 1. 第二型豬環狀病毒(PCV2)感作 32 2. 不同種類LPS處理 32 3. 免疫螢光染色法 33 4. 反轉錄聚合酶鏈鎖反應 33 4-1 RNA之萃取 33 4-2 反轉錄反應 34 4-3 反應溶液成分與條件 35 4-4 聚合酶鏈鎖反應產物電泳 35 5. 病毒力價之測定 36 6. 細胞培養上清液中細胞激素之表現測定 36 7. 統計分析 37 第二節 試管內豬腎細胞株(PK-15)試驗 38 (一)實驗設計及流程圖 38 (二)實驗材料 39 1. 豬腎細胞株(PK-15) 39 2. 細胞培養液 (DMEM-C) 39 3. 細胞清洗液 (DPBS) 39 4. D(+)Glucosamine, Hydrochloride 39 5. Hanks’ Balanced Salt Solutions 39 6. NF-κB activation inhibitor 40 7. Phorbol 12-myristate 13-acetate(PMA) 40 (三)實驗方法 40 1. NF-κB activation inhibitor及不同濃度PMA之處理 40 1-1 第二型豬環狀病毒 (PCV2) 感作 40 1-2  NF-κB activation inhibitor及PMA之處理 41 1-3 細胞形態觀察 41 1-4 病毒力價之測定 41 第四章、結果 43 第一節 感染PCV2豬肺臟巨噬細胞經LPS處理之結果 43 1-1 感染PCV2豬肺臟巨噬細胞經LPS處理前實驗 43 1-1-1  PCV2感染豬肺臟巨噬細胞之形態學觀察 43 1-1-2  豬肺臟巨噬細胞PCV2感染之陽性訊號 43 1-1-3 PCV2感染豬肺臟巨噬細胞之核內陽性率 44 1-2 感染PCV2豬肺臟巨噬細胞經不同濃度L PS處理之結果 50 1-2-1 PCV2感染豬肺臟巨噬細胞之核內陽性率 50 1-3 感染PCV2豬肺臟巨噬細胞經不同來源LPS處理之結果 50 第二節 PCV2複製之中間產物、病毒力價及刺激細胞激素之偵測 53 2-1 免疫螢光染色法 53 2-1-1  PCV2 ORF1 偵測結果 53 2-2 反轉錄聚合酶鏈鎖反應偵測ORF2之結果 53 2-3 病毒力價之測定 54 2-4 細胞培養上清液中細胞激素之表現測定 58 2-4-1  Interleukin (IL) -1β蛋白質之測定 58 2-4-2  Interleukin (IL)-8蛋白質之測定 59 2-4-3  Interleukin (IL)-10蛋白質之測定 59 2-4-4  Tumor necrosis factor alpha(TNF-α)蛋白質之測定 60 第三節 試管內豬腎細胞株(PK-15)試驗 66 3-1 NF-κB activation inhibitor與PMA處理感染PCV2之PK-15細胞株 66 3-1-1 細胞形態觀察之結果 66 3-1-2 病毒力價之結果 66 第五章、討論 70 一、LPS可誘發PCV2於PAMs內進行複製 70 二、LPS與PCV2可協同作用增加細胞激素及細胞動素的表現 75 三、NF-κB活化的抑制可增加PCV2的複製 80 第六章、參考文獻 841700429 bytesapplication/pdfen-US環狀病毒脂多醣lipopolysaccharidePCV2circovirus[SDGs]SDG3以Bacterial lipopolysaccharide活化豬第二型環狀病毒複製之研究Investigation of Porcine Circovirus Type 2 Replication by Bacterial Lipopolysaccharide Activation in vitrothesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59976/1/ntu-95-R93629014-1.pdf