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研究生:黃柏勳
研究生(外文):Bo-Shiun Huang
論文名稱:豬瘟病毒對豬隻周邊血液單核細胞活化分子與interferon-g之影響
論文名稱(外文):The Effects of Classical Swine Fever Virus on the Expression of Activaiton Markers and Interferon-f of Porcine Peripheral Blood Mononuclear Cells
指導教授:李維誠李維誠引用關係
指導教授(外文):Wei-Cheng Lee
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫病理學研究所
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:2002
畢業學年度:90
語文別:中文
中文關鍵詞:豬瘟病毒周邊血液單核細胞活化分子interferon-g
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豬瘟病毒對豬隻免疫系統的傷害為本病主要的致病機制所在。為了探討豬隻的淋巴細胞在豬瘟疫苗或是豬瘟病毒感染後之細胞性免疫之活化狀態,首先利用E2次單位疫苗免疫豬隻,分離周邊血液單核細胞 (peri- pheral blood mononuclear cell;PBMC)進行細胞活化分子CD25與SLA II表現的偵測,結果在免疫後2週內並無法偵測到該兩種分子有明顯表現的情形,另外以豬瘟病毒P.T.株與94.4株進行豬隻之攻毒試驗,免疫組與未免疫之對照組豬隻在攻毒後其PBMC之CD25分子皆無明顯的表現,而SLA II分子的表現在對照組則有明顯被抑制的情形。此外為了探討豬隻在經過E2次單位疫苗或是LPC疫苗免疫之後之細胞性免疫反應,利用純化的豬瘟病毒封套E2醣蛋白進行淋巴增殖試驗,但在實驗結果亦無法偵測到兩組豬隻的PBMC對於該醣蛋白有特異性的增殖反應。T淋巴細胞是免疫系統中相當重要的調控細胞,其所分泌的IFN-g具有調控吞噬細胞與細胞毒殺型T淋巴細胞執行清除細胞內病原的功能,為了探討豬隻在豬瘟病毒感染之後對周邊血液中CD4+與CD8+等T淋巴細胞之IFN-g表現的影響,首先建立以PMA-ionomycin激活淋巴細胞表現IFN-g以及利用細胞內免疫螢光染色配合流式細胞儀偵測的相關技術,之後以豬瘟病毒S59株進行PBMC之in vitro攻毒試驗,結果在未免疫組與LPC免疫組豬隻方面,CD4+與CD8+ T淋巴細胞表現IFN-g的能力受到抑制,而經過E2次單位疫苗免疫兩次的豬隻其CD8+ T淋巴細胞表現IFN-g的平均比例則有增加的情形。
綜合上述結果,豬隻在E2次單位疫苗免疫之後,其PBMC之活化分子CD25與SLA II並無明顯表現的情形,且對於E2醣蛋白亦無特異性的增殖反應,另外豬瘟病毒的感染對於PBMC之SLA II分子的表現以及CD4+與CD8+ T淋巴細胞表現IFN-g的能力有抑制的作用,但是經E2次單位疫苗免疫之後的CD8+ T淋巴細胞表現細胞內IFN-g的平均比例卻有上升的情形。
The impairment of immune system is the major pathogenesis of Classical Swine Fever (CSF). To evaluate the cellular immunity after vaccination and CSF virus (CSFV) infection, peripheral blood mononuclear cells (PBMC) were isolated and submitted for detection of activating markers, CD25 and SLA II, from pigs vaccinated with E2 subunit vaccine. The results indicated that the expression of CD25 and SLA II were not obvious within 2 weeks after vaccination. After challenged with CSFV strain P.T. and 94.4, however, the expression of SLA II in PBMC was more severely suppressed in non-vaccinated pigs compared to vaccinated pigs. Also, the CSFV envelop E2 glycoprotein was used as antigenic stimulant, and the lymphoproliferative responses were undertaken to investigate the CMI response of pigs vaccinated with E2 subunit marker vaccine or LPC vaccine. The results showed that the E2-specific lymphoproliferation of PBMC from vaccinated pigs could not be detected. Alternatively, the effect of CSFV infection on the ability of IFN-g production in CD4+ and CD8+ T lymphocytes was investigated to elucidate the of immune response. PBMC were infected with CSFV strain S59 for 16 hours, and then activated by PMA and ionomycin in the presence of Brefeldin A for 6 hours. IFN-g production was detected by intracellular immunofluorescent staining using flow cytometry. The results indicated that the IFN-g producing cells in CD4+ and CD8+ T lymphocytes were decreased in pigs that were not vaccinated and vaccinated with LPC vaccine. However, the IFN-g producing cells in CD8+ T lymphocytes were increased in pigs vaccinated with E2 subunit vaccine. Taken these data together, it was concluded that the expression of activation marker, CD25 and SLA II, and the proliferation to E2 glycoprotein of PBMC in pigs vaccinated with E2 subunit vaccine and LPC vaccine were not evident. However, the ability of CD4+ and CD8+ T lymphocytes producing IFN-g in non-vaccinated pigs may be impaired by CSFV, but this may not been seen in pigs vaccinated with E2 subunit marker vaccine.
中文摘要 ………………………………………………………………....I
英文摘要 …………………………………………………………………II
目錄 ………………………………………………………………………IV
第一章 緒言 …………………………………………………………….1
第二章 文獻探討 ……………………………………………………….3
2.1 輔助型T淋巴細胞細胞素之免疫調控功能 …………………3
2.2 豬瘟病毒 ………………………………………………………11
第三章 豬隻淋巴細胞內IFN-g表現之偵測 …………………………..17
3.1 前言 ……………………………………………………………17
3.2 實驗設計 ………………………………………………………19
3.3 材料與方法 ……………………………………………………19
3.4 結果 ……………………………………………………………22
3.5 討論 ……………………………………………………………25
第四章 豬瘟病毒對於豬隻淋巴細胞之活化分子
與IFN-g表現之影響 …………………………………………...43
4.1 前言 ……………………………………………………………43
4.2 實驗設計 ………………………………………………………44
4.3 材料與法 ………………………………………………………45
4.4 結果 ……………………………………………………………..51
4.5 討論 ……………………………………………………………..53
參考文獻 ………………………………………………………………….71
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