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研究生:陳憶平
研究生(外文):IPing Chen
論文名稱:豬生殖與呼吸綜合症病毒感染對豬肺泡巨噬細胞細胞激素表現之影響
論文名稱(外文):The cytokine expression in porcine alveolar macrophages after porcine reproductive and respiratory syndrome virus infection
指導教授:鄭謙仁龐飛龐飛引用關係
指導教授(外文):Jeng Chian-RenPang Victor-Fei
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
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:2001
畢業學年度:89
語文別:中文
論文頁數:99
中文關鍵詞:豬生殖與呼吸綜合症病毒細胞激素
外文關鍵詞:porcine reproductive and respiratory syndrome viruscytokine
相關次數:
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豬生殖與呼吸綜合症病毒 (porcine reproductive and respiratory syndrome virus; PRRSV) 歸類於動脈病毒科,為一正向單股具封套的RNA病毒。本病毒主要造成豬隻繁殖障礙及呼吸道疾病,罹病保育豬隻常因二次性細菌感染死亡,而導致養豬業者重大的經濟損失;至今本病毒對免疫系統的影響仍未完全清楚。由於此病毒主要對單核吞噬細胞具有親合力,特別是肺泡巨噬細胞 (porcine alveolar macrophage; PAM),因此本研究以M.O.I.為0.1的MD001 PRRSV分離株活體外感染PAM,在感染6-48小時觀察PAM的存活率、細胞凋零率、感染率及細胞激素之變化情形,希望由感染PRRSV的PAM之表現,進而了解病毒感染是否會傷害及干擾免疫協調系統。結果發現感染病毒的PAM在6小時即可偵測到病毒的抗原及核酸,雖然PAM維持於約6.4 % 的低感染率,但細胞存活率在攻毒後24小時顯著下降至55.0 %,在攻毒後48小時持續維持於49.0 %。PAM細胞凋零率在攻毒後12小時顯著增加至18.2 %,且在攻毒後48小時持續增加至41.4 %。以建立的同步定量聚合酶鏈鎖反應偵測 PAM細胞激素TNF-a、IL-1b、IL-8 mRNA的表現,於攻毒6-24小時相對於對照組分別增加了3.5-76.6倍、8.6-58.8倍、1.2-19.7倍,但至攻毒48小時後與對照組則無顯著差異。IL-18在攻毒後6小時呈增加的趨勢,但其後開始下降至攻毒後48小時相對於對照組顯著減少了 6.5倍。以ELISA偵測TNF-a及IL-1b的蛋白質表現,TNF-a於攻毒後6-24小時呈增加趨勢,而IL-1b於攻毒後6小時亦呈增加的趨勢,到攻毒後24-48小時相較於對照組更呈顯著的增加。由上述結果得知PRRSV感染後除了可傷害PAM造成存活率的降低及細胞凋零的產生外,對細胞激素TNF-a、IL-1b及IL-8尚具提升作用。就存活的個別細胞而言,PRRSV可顯著提升這些細胞激素的表現及分泌,此可能與PRRSV感染後造成的發燒與間質性肺炎有關。相對於炎症細胞激素的提升,與細胞性免疫有關的IL-18於PRRSV感染後則呈抑制的現象,暗示病毒或許可以減緩細胞性免疫而使其易在宿主中建立初期感染。而就PRRSV如何以低感染率卻造成顯著的細胞傷害及免疫功能干擾,則仍待進一步探討。
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, positive-stranded RNA virus classified in the Arteriviridae family. The disease causes by PRRSV infection in pigs is characterized by severe reproductive failure and interstitial pneumonia. PRRSV infection in young pigs is frequently complicated with secondary infection, which may cause severe economic loss in pig industry. Until now, the impact of virus infection in immune system is not well understood. Due to monocyte/macrophage lineage, especially porcine alveolar macrophage (PAM) is the major permissive cell for PRRSV replication. To verify the role of PRRSV infection in porcine immune system, alterations of viability, apoptosis, infectious rate, and cytokines expression and secretion in PAM were evaluated in vitro for 6-48 hours post inoculation (HPI). The virus strain used was MD001, and the dose was 0.1 multiplicity of infection (M.O.I.). The result showed viral protein and nucleic acid could be detected in PAM as early as 6 hours after infection, however, a consistent low infectious rate about 6.4 % were present throughout the study. When compared with a mock-infected group, PAM from the PRRSV infected group had a significant low viability from 55.0 % to 49.0 % at 24-48 HPI and a significant high apoptotic rate from 18.2 % to 41.4 % at 12-48 HPI. The mRNA levels of TNF-a, IL-1b, and IL-8 were increased 3.5-76.6 fold, 8.6-58.8 fold, and 1.2-19.7 fold separately in PRRSV infected PAM during 6-24 HPI by real-time PCR. The mRNA expression of IL-18, however was decreased 6.5 fold in PRRSV infected PAM at 48 HPI. The protein levels of TNF-a and IL-1b were increased in PRRSV infected PAM, and significant increased was present in IL-1b at 24-48 HPI compared to mock control. The profound expression and secretion of proinflammtory cytokines associated with cell injury in PRRSV infected PAM may correlate with clinical signs of pyrexia and interstitial pneumonia. In addition, inhibition of IL-18 expression may reduce cell-mediated immune activity and contribute to early infection of PRRSV. The possible mechanism in low infectious rate associated with profound impact of cell injury and immune irregulation in PRRSV infection still need to be elucidated.
目錄
第一章緒言-------------------------------------------------1
第二章文獻探討---------------------------------------------3
第一節豬生殖與呼吸綜合症-----------------------------------3
一、歷史背景及經濟重要性------------------------------3
二、病毒特性------------------------------------------5
三、臨床症狀及病理變化--------------------------------6
四、致病機制------------------------------------------9
第二節豬生殖與呼吸綜合症對免疫系統的影響------------------13
一、對體液及細胞性免疫之影響-------------------------13
二、對細胞激素表現之影響-----------------------------17
第三節 同步定量聚合脢鏈鎖反應------------------------------21
第三章材料與方法------------------------------------------25
第一節實驗材料--------------------------------------------25
一、豬生殖與呼吸綜合症病毒---------------------------25
二、細胞清洗液 (RPMI-W)------------------------------26
三、細胞培養液 (RPMI-C)------------------------------26
四、豬肺泡巨噬細胞之採集-----------------------------26
五、致裂原-------------------------------------------27
第二節實驗設計及流程圖------------------------------------28
第三節實驗方法--------------------------------------------29
一、存活率的測定-------------------------------------29
二、TUNEL assay--------------------------------------29
三、免疫螢光染色法-----------------------------------30
四、同步定量聚合脢鏈鎖反應---------------------------31
1.樣品之收集與製備---------------------------------31
(1) RNA之萃取-------------------------------------31
(2) 反轉錄反應------------------------------------33
2.引子與探針之設計---------------------------------33
3.反應溶液成分與條件-------------------------------36
4.聚合脢鏈鎖反應產物電泳與定序---------------------36
5.資料分析-----------------------------------------37
6.增幅效率差異之檢測-------------------------------38
五、細胞激素蛋白質表現之測定-------------------------38
1.TNF-a蛋白質表現之測定----------------------------38
2.IL-1b蛋白質表現之測定----------------------------39
第四節統計方法--------------------------------------------41
第四章結果------------------------------------------------43
第一節 建立同步定量聚合脢鏈鎖反應之分析方法----------------43
一、聚合脢鏈鎖反應產物電泳與定序分析-----------------43
二、各細胞激素及病毒量與G3PDH增幅效率的差異----------43
第二節豬生殖與呼吸綜合症病毒感染對豬肺泡巨噬細胞之影響----52
一、存活率-------------------------------------------52
二、細胞凋零率---------------------------------------53
三、感染率-------------------------------------------54
四、細胞激素及病毒量之表現---------------------------54
1.對IL-1b mRNA 表現量之影響------------------------55
2.對IL-8 mRNA 表現量之影響-------------------------55
3.對TNF-a mRNA表現量之影響-------------------------56
4.對IL-18 mRNA 表現量之影響------------------------57
5.對病毒表現量之影響-------------------------------58
6.TNF-a蛋白質之表現--------------------------------59
7.IL-1b蛋白質之表現--------------------------------60
第五章 討論------------------------------------------------75
第一節偵測方法之檢討--------------------------------------75
第二節豬生殖與呼吸綜合症病毒感染對豬肺泡巨噬細胞之影響----77
第六章 參考文獻--------------------------------------------87
表次
表 3. 1豬隻細胞激素及病毒的引子及探針----------------------35
圖次
圖 2. 1-1豬生殖與呼吸綜合症病毒可能的致病機序----------------12
圖 2. 2-1同步定量聚合脢鏈鎖反應的偵測原理--------------------23
圖 2. 2-2同步定量聚合脢鏈鎖反應的增幅圖----------------------24
圖 3. 1 豬隻肺沖洗液細胞中肺泡巨噬細胞所佔的比率------------42
圖 3. 2 豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,以免 疫螢光染色法觀察其感染率-------------------------------------42
圖 4. 1-1聚合脢鏈鎖反應產物電泳圖----------------------------45
圖 4. 1-2G3PDH總RNA濃度的對數值與CT值間的線性圖--------------46
圖 4. 1-3IL-1b與G3PDH之間增幅效率的差異----------------------47
圖 4. 1-4IL-8與G3PDH之間增幅效率的差異-----------------------48
圖 4. 1-5IL-18與G3PDH之間增幅效率的差異----------------------49
圖 4. 1-6TNF-a與G3PDH之間增幅效率的差異----------------------50
圖 4. 1-7PRRSV與G3PDH之間增幅效率的差異----------------------51
圖 4. 2-1豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,細胞存活率之變化-------------------------------------------------61
圖 4. 2-2豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,細胞凋零之情形---------------------------------------------------62
圖 4. 2-3豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,細胞凋零率之變化-------------------------------------------------66
圖 4. 2-4豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,感染率之情形-----------------------------------------------------67
圖 4. 2-5豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,IL-1b mRNA表現量之改變------------------------------------------68
圖 4. 2-6豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,IL-8 mRNA表現量之改變-------------------------------------------69
圖 4. 2-7豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,TNF-a mRNA表現量之改變-------------------------------------------70
圖 4. 2-8豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,IL-18 mRNA表現量之改變------------------------------------------71
圖 4. 2-9豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,PRRSV RNA表現量之改變----------------------------------------72
圖 4. 2-10豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,TNF-a蛋白質表現量之改變------------------------------------------73
圖 4. 2-11豬肺泡巨噬細胞活體外感染豬生殖與呼吸綜合症病毒後,IL-1b蛋白質表現量之改變-----------------------------------------74
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