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研究生:高啟霏
研究生(外文):Chi-Fei Kao
論文名稱:疫苗免疫下攜帶不同病毒量豬第二型環狀病毒亞臨床豬隻單核球來源巨噬細胞功能之比較
論文名稱(外文):Comparisons on the function of monocyte-derived macrophages (MDMs) of vaccinated subclinically PCV2-infected pigs with different viral loads
指導教授:龐飛龐飛引用關係
指導教授(外文):Victor Fei Pang
口試委員:張惠雯劉涓張志成
口試委員(外文):Hui-wen ChangJiuan Judy LiuChih-Cheng Chang
口試日期:2015-06-10
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子暨比較病理生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:84
中文關鍵詞:豬第二型環狀病毒亞臨床感染病毒量單核球來源巨噬細胞豬環狀病毒相關性疾病疫苗
外文關鍵詞:PCV2subclinicalviral loadMDMsPCVADvaccine
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豬第二型環狀病毒(PCV2)為引起離乳後多系統消耗性症候群(PMWS)和一系列豬環狀病毒相關性疾病(PCVAD)的主要致病原。近年來,疫苗施打的普及已成功降低了感染後的發病率,大多數豬隻在感染PCV2後發展為不具臨床症狀且僅含少量血清及組織病毒量的亞臨床感染。目前已知,PCV2感染淋巴球及單核球類白血球並可影響其功能,然而唯有輔助因子的共同存在,所引起之免疫失調才能造成血清與組織內病毒量的過度增加,促使臨床症狀產生。儘管離乳後多系統消耗性症候群的特徵性病變,肉芽腫性炎症反應,反應了巨噬細胞的參與,現有針對PMWS發病豬或亞臨床感染豬隻體內巨噬細胞與PCV2交互作用進行探討的研究仍十分有限。本研究依據血清病毒量中位數,將已經疫苗免疫之PCV2亞臨床感染豬隻分成高與低病毒組,並利用脂多醣(LPS)及/或PCV2攻毒,模擬豬隻在田間受革蘭氏陰性菌感染或再次暴露於PCV2的環境,來探討不同病毒量之PCV2亞臨床感染豬隻單核球來源巨噬細胞(MDMs)的功能變化。由於實驗期間,採樣豬場曾更換PCV2的免疫計劃,因此在結果分析時,將一併比較前後使用之FosteraTM PCV和Circovac®兩種類型疫苗造成MDMs功能影響的差異。整體而言,於三週齡施打FosteraTM PCV的豬隻多半具有較低的血清病毒量。來自高病毒組豬隻的MDMs通常會表現較高的存活率並含有較高的細胞內病毒量。在細胞激素方面,可以發現LPS和PCV2對於誘導IL-1β、IL-12、TNF-α及IFN-α mRNA表現具有明顯的協同效果;在各處理組可見當豬隻病毒量越高,MDMs IL-10的表現越低但IL-12的表現卻越高,兩者呈相反的走勢;同屬趨炎症型細胞激素之TNF-α和IFN-α的表現則類似IL-12。至於MDMs的吞噬能力則和病毒量之間有明顯的負相關。不同疫苗對MDMs的功能影響大致與病毒量的影響相符。依據研究結果推測,具有較高病毒量的PCV2亞臨床豬隻,其體內MDMs可能傾向引起過度的淋巴球活化而有助於PCV2增殖;大量的趨炎症型細胞激素分泌則可能會導致較劇烈炎症反應及組織傷害;至於吞噬能力的減弱,將不利於MDMs執行微生物殺滅、抗原呈現及組織修復等能力,並提高豬隻對繼發感染的感受性。因此,縱使PCV2亞臨床豬隻外觀看似健康,仍有發展為PCVAD的風險,特別是攜帶較高病毒量的豬隻。本研究所觀察到之PCV2病毒量對MDMs功能的影響,或許為PCV2的致病機制帶來了另一新的觀點。

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated diseases (PCVAD), including postweaning multisystemic wasting syndrome (PMWS), and is considered to play a necessary but insufficient role in the entire pathogenesis. Recently, the widespread vaccination program has significantly decreased the prevalence of clinical diseases and the asymptomatic PCV2 infection with low serum and tissue viral loads, known as subclinical infection, dominates the post-infection outcome. It is known that PCV2 targets mononuclear leukocytes, including lymphocytes and monocyte/macrophage lineage cells, and can affect their immune functions. However, only with the help of various co-factors, the immune dysfunction is able to cause excessive increase in serum and tissue viral loads, and subsequent clinical disease development. The granulomatous lesion, a hallmark of PMWS, highlights the participation of macrophages, yet information is limited in disclosing the interaction between PCV2 and infected macrophages, no matter in PMWS-affected or subclinically infected pigs. Hence, by categorizing pigs into higher (HV) or lower (LV) viral load groups based on the medium value of serum viral loads, we investigated the functional differences in MDMs of vaccinated subclinically PCV2-infected pigs carrying different viral loads following incubation with lipopolysaccharide (LPS) and/or PCV2 in vitro to mimic field conditions of Gram-negative bacterial infection and/or PCV2 superinfection. Besides, due to the change of vaccination program in the pig farm during the course of study, we also compared the functional differences in MDMs using different vaccines, FosteraTM PCV and Circovac®. In general, pigs receiving FosteraTM PCV at 3 weeks of age tended to have lower serum viral loads. The HV MDMs were prone to have relatively higher survival rate and viral loads. There was an apparent synergism of concurrent PCV2 and LPS treatment on the mRNA expression levels of IL-1β, IL-12, TNF-α, and IFN-α. The trend of IL-10 and IL-12 expression in HV and LV pigs was controversy, with the higher viral load the lower IL-10 and the higher IL-12 expression regardless of the treatment. The pro-inflammatory cytokines TNF-α and IFN-α displayed similar expression pattern as seen in IL-12. A significantly reduced phagocytotic activity was observed in HV group. The effect of vaccination program on the functions of MDMs was in close association with the resulted serum viral load. Based on the results, it is speculated that, for those pigs carrying higher viral load, the activation of lymphocytes evoked by the lower IL-10 and higher IL-12 expressed by MDMs may potentiate improper immune activation in favor of PCV2 replication. The relatively higher expression of TNF-α and IFN-α may aggravate tissue damage due to excessive inflammation modulated by MDMs. Moreover, the higher survival rate but impaired phagocytosis of MDMs may compromise the ability of microbial killing, antigen presenting, and tissue repair which may predispose those pigs to secondary infection. Thus, despite the healthy look, it is worth noting that the subclinically PCV2-infected pigs, especially those carrying higher viral loads, may be still in danger of developing clinical diseases under the help of other co-factors. Furthermore, the functional alteration discovered in MDMs under different viral loads may provide another point of view toward understanding the pathogenesis of PCV2.

目錄
口試委員審定書................................................................................................................i
誌謝...................................................................................................................................ii
中文摘要..........................................................................................................................iii
英文摘要...........................................................................................................................v
目錄.................................................................................................................................vii
表次...................................................................................................................................x
圖次..................................................................................................................................xi
縮寫對照表....................................................................................................................xiii
第一章 序言…………………………………………………………………………...1
第二章 文獻回顧……………………………………………………………………...3
第一節 豬環狀病毒………………………………………………………………...3
1.1豬環狀病毒概述……………………………………………………………….3
1.2豬環狀病毒之傳播……………………………………………….....................4
第二節 豬環狀病毒相關性疾病 (Porcine Circovirus-Associated Diseases; PCVAD)………………………………………………………………………….……5
2.1豬環狀病毒相關性疾病之臨床症狀與組織病變………………………….5
2.2豬環狀病毒相關性疾病之致病機制與發病因子………………………….6
2.3 豬環狀病毒相關性疾病之診斷與免疫計畫………………………………8
第三節 豬環狀病毒之複製………………………………………………………...9
第四節 豬第二型環狀病毒對免疫系統之影響………………………………….11
4.1豬第二型環狀病毒量對感染豬隻的影響………………………………...11
4.2豬第二型環狀病毒對淋巴系統之影響…………………………………...12
4.3豬第二型環狀病毒對細胞激素分泌之影響……………………………...13
4.4豬第二型環狀病毒對先天免疫(innate immunity)之影響………………..15
4.5豬第二型環狀病毒亞臨床感染…………………………………………...16
第三章 材料與方法………………………………………………………………….19
第一節 實驗設計………………………………………………………………….19
第二節 實驗材料………………………………………………………………….21
2.1實驗動物與血液樣本………………………………………………………...21
2.2細胞培養基及相關試劑……………………………………………………...22
2.3核酸抽取試劑………………………………………………………………...24
2.4即時聚合酶鏈鎖反應之商品化試劑………………………………………...25
2.5流式細胞儀相關試劑………………………………………………………...27
2.6儀器…………………………………………………………………………...28
第三節 實驗方法………………………………………………………………….28
3.1單核球來源巨噬細胞之製備………………………………………………...28
3.2血漿、細胞及上清液之豬第二型環狀病毒核酸抽取……………………...31
3.3細胞RNA抽取……………………………………………………………….31
3.4反轉錄聚合酶鏈鎖反應……………………………………………………...32
3.5利用即時聚合酶鏈鎖反應檢測第二型豬環狀病毒病毒量及細胞激素
(IL1-β、IL-10、IL-12、TNF-α、IFN-α) mRNA的表現…………………...32
3.6以Propidium iodide (PI)染色檢測細胞存活率……………………………...33
3.7以商品化試劑pHrodo® dye檢測細胞吞噬能力…………………………....34
3.8統計分析…………………………………………………………………...…34
第四章 結果………………………………………………………………………….36
第一節 樣本收集與細胞純化…………………………………………………….36
1.1 樣本收集……………………………………………………………………..36
1.2 PBMCs與MDMs的純化…………………………………………………...36
第二節 細胞存活率……………………………………………………………….37
2.1不同病毒量之MDMs在給予不同處理後存活率的差異………………….37
2.2以不同疫苗免疫之MDMs在給予不同處理後存活率的差異…………….38
第三節 細胞病毒量變化………………………………………………………….38
3.1不同病毒量之MDMs在給予不同處理後病毒量之變化…………………..39
3.2以不同疫苗免疫之MDMs在給予不同處理後病毒量之變化……………..39
第四節 細胞激素mRNA的變化…………………………………………………40
4.1不同病毒量之Mock組MDMs各細胞激素mRNA表現量…………….....41
4.2不同病毒量之MDMs在給予不同處理後細胞激素mRNA表現量的差異
………………………………………………………………………………...41
4.3以不同疫苗免疫之MDMs在給予不同處理後細胞激素mRNA表現量的差
異……………………………………………………………………………...43
第五節 MDMs吞噬能力的變化……………………………………………….....45
5.1不同病毒量之MDMs在給予不同處理後吞噬能力之差異……………......45
5.2以不同疫苗免疫之MDMs在給予不同處理後吞噬能力之差異..................45
第五章 討論.................................................................................................................59
第六章 參考文獻.........................................................................................................73


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