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研究生:駱正峰
研究生(外文):Cheng-Feng Lo
論文名稱:確認並分析巨細胞病毒磷酸化蛋白65-3在正常老鼠所誘發的自體免疫反應和人類病理現象的相似性
論文名稱(外文):Identification and characterization of pp65-3 induced autoimmunity in normal strain of mice and their similarity in human patients
指導教授:張銘一
指導教授(外文):Minigi Chang
學位類別:碩士
校院名稱:慈濟大學
系所名稱:微免暨分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:94
語文別:中文
論文頁數:56
中文關鍵詞:紅斑性狼瘡巨細胞病毒腎絲球腎炎自體抗體
外文關鍵詞:SLECMVglomerulonephritisautoantibody
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自體免疫疾病的發病機轉至今仍不是十分清楚,但是大致上可分為三個原因。環境因素、遺傳背景和免疫調控。根據我們實驗室之前的研究發現,在台灣,86%的SLE病人可以測得到HCMV pp65的抗體。此外、動物模式顯示出Balb/c和C57BL/6在pp65-3免疫過後會發展出和Hela cell抗原交叉反應的抗體。很有趣的一點是,由SLE病人血清中純化出來的pp65-3抗體和老鼠pp65-3的抗體所辨識的Hela cell抗原極為相似。由血清學的證據可以推測,免疫過後的老鼠,其免疫耐受性有破壞的現象,但尚未發展出臨床症狀。由最近的證據顯示出,HCMV pp65-3的抗原決定位,可能具有誘發tolerance break的能力。然而HCMV pp65在致病上,所扮演的角色,仍不清楚。
這個計畫的目的是確認並分析pp65-3所誘發的自體免疫反應,設法找出會和pp65抗體反應的自體抗原。此外,利用免疫螢光染色以及蘇木紫染色對pp65-3免疫後的老鼠摘取腎臟加以觀察,是否有發炎以及淋巴球浸潤。接著,在研究病人交叉反應抗體效價,以及和SLE疾病發病的關聯性。
The etiopathology of the autoimmune diseases are not well understood. Both environmental triggers and genetic background have been postulated as possible causes for autoimmunity. According to our prior studies, we found that over 86% of SLE patients from Taiwan developed antibody to HCMV pp65 antigen. In addition, animal model showed that Balb/c and C57/B6 mice developed crossed-reactive antibodies to Hela antigen following immunization of purified HCMV pp65-3 antigen . It is interesting to note that affinity purified anti-pp65 antibody from SLE patients also react to similar antigen identified by immunized mouse sera. In the immunoprecipitation and western assays, sera of pp65-3 immunized mice reactive against antigens that were immunoprecipited by human SLE patients. Regardless significant serological data suggested a tolerance break by immunized normal strains, none of these animals developed clinical symptoms. Current results imply that HCMV pp65-3 peptide contains epitopes that is capable to induce tolerance break. However, the pathogenesis potential for HCMV pp65-3 is not yet evaluated.
This project is designed to identify and characterized the autoimmunity induced by pp65-3 peptide.Moreover, sera and tissue sections from immunized animals were analyzed by immunostain and HE to verify signs of inflammation and infiltrated lymphocytes. The titer of cross-reactive antibody by patients were studied and compared to elucidate the connection to clinical manifestation.
目錄
中文摘要----------------------------------------------------Ⅲ
英文摘要----------------------------------------------------Ⅳ
中英文對照表----------------------------------------------Ⅴ
第一章 序論----------------------------------------------- 1
第一節:系統性紅斑性狼瘡--------------------------- 1
一、系統性紅斑性狼瘡病症--------------------- 1
二、系統性紅斑性狼瘡發病成因--------------- 2
三、SLE相關的自體抗體------------------------- 3
四、SLE自體抗原的分子量大小---------------- 4
五、SLE誘發的腎絲球腎炎---------------------- 4
第二節:巨細胞病毒及自體免疫--------------------- 5
一、巨細胞病毒----------- ------------------------- 5
二、巨細胞病毒磷酸化蛋白65------------------- 6
三、巨細胞病毒pp65-3基因圖譜簡介-----------7
第三節:模式動物---------------------------------------- 7
第四節:研究動機---------------------------------------- 8
第二章:材料與方法-------------------------------------10
第一節:實驗材料----------------------------------------10
一、細胞株------------------------------------------- 10
二、實驗動物---------------------------------------- 10
三、血清樣本---------------------------------------- 10
四、使用的抗體及重組蛋白---------------------- 10
五、相關藥品---------------------------------------- 11
第二節:實驗方法--------------------------------------- 12
一、蘇木紫染色?------------------------------------- 12
二、免疫螢光染色----------------------------------13
三、間接免疫螢光染色法-------------------------13
四、 西方墨點法------------------------------------14
五、蛋白質純化------------------------------------ 14
六、PP65樹酯製備--------------------------------- 15
七、血清抗體純化----------------------------------15
八、動物免疫----------------------------------------15
第三章 結果------------------------------------------------16
第四章 討論------------------------------------------------20
第五章 結論------------------------------------------------25
第六章 結果圖---------------------------------------------26
附錄一------------------------------------------------------ 45
第七章 參考文獻----------------------------------------- 46
第七章 參考文獻

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