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研究生:洪子涵
研究生(外文):Zih-Hang Hung
論文名稱:假性狂犬病毒誘發巨噬細胞發炎反應之研究
論文名稱(外文):Study of the cellular inflammatory response of macrophage induced by pseudorabies virus infection
指導教授:張天傑
指導教授(外文):Tien-Jye Chang
口試委員:徐維莉張元衍
口試日期:2013-07-11
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
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:2013
畢業學年度:101
語文別:中文
論文頁數:78
中文關鍵詞:假性狂犬病毒巨噬細胞氧化壓力抗氧化
外文關鍵詞:pseudorabies virusmacrophageoxidative stressanti-oxidant
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假性狂犬病毒(Pseudorabies virus, PRV)屬於皰疹病毒科的α-皰疹病毒亞科,具有封套的雙股DNA病毒,是造成假性狂犬病(pseudorabies, PR)的致病原。假性狂犬病毒可感染的宿主範圍很廣,大多數的哺乳類動物皆可受到感染,但並不感染人類。假性狂犬病會因豬隻年齡大小而出現不同的症狀,包括:兩週齡以下的小豬感染後死亡率可接近100%、懷孕母豬的流產、成豬感染後造成呼吸道及生殖系統上的疾病,因而造成養豬場重大的經濟損失。先前本實驗室研究發現假性狂犬病毒感染RAW264.7小鼠巨噬細胞後給予抗氧化物質可降低發炎反應的發生,是否病毒感染後誘發細胞內ROS上升而使病毒感染受到影響,目前仍不清楚。因此,本研究將探討假性狂犬病毒(PRV)引起細胞發炎反應及氧化壓力變化之研究。
本實驗首先觀察PRV感染後ROS(reactive oxygen species)變化,使用0.1MOI之PRV感染劑量在感染6小時、12小時及24小時後,不論利用Griess reagent測定一氧化氮(NO)濃度,還是以西方點墨法觀察iNOS蛋白質表現量,結果皆顯示感染24小時有大量NO產生,同時亦可觀察到細胞內有過氧化脂質(MDA)堆積的現象(p<0.05)。以ELISA分析結果發現PRV感染時會產生大量IL-6、MCP-1、TNF-α (p<0.05),並影響信息傳遞路徑:NF-κB、mitogen-activated protein kinase(MAPK)路徑及JAK/STAT路徑的蛋白質表現。此外,為了瞭解ROS產生的途徑,利用西方墨點法觀察NADPH oxidase、heme oxygenase-1(HO-1)、AMPK及Nrf-2磷酸化表現情形,結果顯示PRV感染後皆可增加其表現量。另一方面,為了證實PRV感染會調控細胞內抗氧化系統,測定抗氧化酵素SOD、catalase及GPx的酵素活性,並以西方墨點法觀察其蛋白質表現量變化,結果發現PRV感染可增加抗氧化酵素SOD、catalase及GPx的表現。本研究結果顯示假性狂犬病毒感染對於巨噬細胞的影響可能與ROS及若干轉錄因子的活化有關,有助於未來了解假性狂犬病毒感染造成發炎反應機轉的研究。


Pseudorabies virus (PRV), a member of the alpha-herpesvirinae subfamily in the family Herpesviridae, is the causative agent of Aujeszky’s disease (AD, pseudorabies). PRV has a broad host range infecting most mammals except humans. The severity of clinical signs depends on the age of the pig. Young piglets are highly susceptible with mortality rates reaching 100% during the first 2 weeks of age. Infected adult pig shows primarily respiratory and reproductive diseases. In previous study, antioxidants can regulate PRV-induced inflammatory response in RAW264.7 cells. The relationship between virus infection and ROS (reactive oxygen species) production is still unclear. In this study, we investigated the inflammatory response and the status of oxidant/antioxidant system in PRV-infected RAW264.7 cells. To investigate intracellular ROS production induced by PRV, RAW264.7 cells were infected with PRV at 0.1 multiplicity of infection for 6 hours, 12 hours and 24 hours. Results indicated a significant increase of NO, TNF-α, IL-6, and MCP-1(p<0.05), and an increased level of NF-κB, ERK, p38 and JNK MAPKs in PRV-infected RAW264.7 cells. Moreover, malondialdehyde (MDA), an oxidative stress marker, is also increased(p<0.05). Western blotting showed that NADPH oxidase, heme oxygenase-1, AMPK and Nrf2 level were up-regulated. Our data also showed that PRV infection induced antioxidant enzyme response, such as SOD, catalase and GPx. In conclusion, ROS production and several transcription factor obviously play important roles on the inflammatory responses in PRV-infected RWA264.7 cells.

中文摘要................................................. I
Abstract............................................... III
目錄 ................................................IV
圖表次 ................................................VII
第一章、 緒言 .........................................1
第二章、 文獻探討 .........................................2
第一節 假性狂犬病(pseudorabies, PR)簡介 .................2
第二節 假性狂犬病毒簡介 .................................2
第三節 假性狂犬病毒的致病機轉 .................................5
第四節 假性狂犬病臨床症狀與病理變化 .........................5
第五節 宿主細胞的氧化/抗氧化平衡機制 .........................6
一、 ROS(reactive oxygen species)及RNS(reactive nitrtogen species)的產生 .........................................6
二、 產生ROS的相關酵素 .................................7
三、 一氧化氮(NO) ........................................10
四、 ROS啟動細胞訊息傳遞 ................................10
五、 細胞內氧化壓力(Oxidative stress)的形成與抗氧化系統....... 11
第三章、材料與方法 ........................................15
第一節 細胞培養 ........................................15
第二節 冷凍細胞 ........................................15
第三節 解凍細胞 ........................................16
第四節 病毒株 ........................................16
第五節 病毒增殖 ........................................16
第六節 病毒斑試驗 ........................................17
第七節 細胞型態觀察 ........................................17
第八節 蛋白質分析 ........................................17
一、 細胞樣本收集 ........................................17
二、 細胞蛋白質濃度測定.....................................18
三、 細胞蛋白質萃取 ........................................18
第九節 蛋白質電泳分析...................................... 18
一、 SDS-PAGE製備 ........................................18
二、 蛋白質電泳 ........................................18
三、 西方墨點法(Western blot)..............................19
四、 冷光壓片 ........................................19
五、 蛋白質表現量分析 ................................19
第十節 細胞內乳酸脫氫酶(lactate dehydrogenase, LDH)濃度測定....20
第十一節 一氧化氮(NO)濃度測定.................................20
第十二節 以酵素連結免疫吸附分析法(Enzyme-linked immunosorbent assay, ELISA)測定IL-6, TNF-α, MCP-1濃度變化................20
第十三節 細胞內Malondialdehyde (MDA, 丙二醛)濃度測試...........21
第十四節 細胞內抗氧化酵素測定 .................................21
一、 Superoxidase Dismutase (SOD).........................21
二、 Catalase(CAT) .................................22
三、 Glutathione peroxidase (GPx): .................22
第十五節 統計分析 .........................................23
第四章、 結果 .........................................24
第一節 以顯微鏡觀察假性狂犬病毒(PRV)感染小鼠巨噬細胞(RAW264.7)之細胞型態變化 .................................................24
第二節 假性狂犬病毒對細胞毒性之影響–乳酸脫氫酶活性測定.............24
第三節 在不同時間點觀察假性狂犬病毒感染對RAW264.7細胞產生一氧化氮(NO)之影響 .................................................25
第四節 以西方墨點法觀察假性狂犬病毒對於RAW264.7細胞內iNOS及COX2蛋白質表現之影響 .........................................25
第五節 以ELISA分析假性狂犬病毒感染後誘發RAW264.7細胞促發炎性細胞激素(pro-inflammatory cytokine)產生之影響 .................25
第六節 以西方墨點法觀察假性狂犬病毒感染對於RAW264.7細胞內NF-κB蛋白質表現之影響 .................................................26
第七節 以西方墨點法觀察假性狂犬病毒感染對於RAW264.7細胞內MAPK路徑蛋白質表現之影響 ........................................26
第八節 以西方墨點法觀察假性狂犬病毒感染對於RAW264.7細胞內JAK/STAT路徑蛋白質表現之影響 .........................................27
第九節 假性狂犬病毒感染造成RAW264.7細胞內丙二醛(malondialdehyde, MDA)濃度大量增加 .........................................28
第十節 假性狂犬病毒感染後對RAW264.7細胞內NADPH oxidase的活化....28
第十一節 假性狂犬病毒感染對於RAW264.7細胞內AMPK蛋白質表現之影響...29
第十二節 假性狂犬病毒感染對RAW264.7細胞內SOD酵素活性及蛋白質表現之影響 .........................................................29
第十三節 假性狂犬病毒感染對RAW264.7細胞內catalase酵素活性及蛋白質表現之影響 .................................................30
第十四節 影響假性狂犬病毒感染對RAW264.7細胞Nrf2、HO-1蛋白質表現量之影響 .................................................30
第十五節 假性狂犬病毒感染對RAW264.7細胞內GPx酵素活性之影響.......31
第五章、 討論..............................................62
參考文獻 .................................................68


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