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系統識別號 U0026-0812200911432902
論文名稱(中文) 上皮細胞之膜上蛋白在嚴重急性呼吸系統症候群自身免疫反應之研究
論文名稱(英文) The study of the epithelial cell membrane protein in SARS autoimmunity
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 93
學期 2
出版年 94
研究生(中文) 馮台雲
研究生(英文) Tai-Yun Feng
電子信箱 seahorse_323@hotmail.com
學號 T9692104
學位類別 碩士
語文別 中文
論文頁數 71頁
口試委員 口試委員-林以行
指導教授-郭余民
口試委員-廖寶琦
中文關鍵字 嚴重急性呼吸系統症候群  自體免疫反應  甘油醛-3-磷酸脫氫酶 
英文關鍵字 SARS  autoimmunity  GAPDH 
學科別分類
中文摘要   嚴重急性呼吸系統症候群(SARS),是一種由非典型冠狀病毒感染所造成的病毒性肺炎。而肺臟是受到SARS此疾病所影響最主要的器官。另外,由病毒感染引起肺泡上皮細胞和微血管內皮細胞嚴重的傷害,會造成肺、肺泡間質組織以及肺泡隔膜的纖維化,最後導致肺部纖維化和肺膨脹不全的症狀。由纖維化所造成呼吸道功能逐漸衰退,最後可能會造成患者死亡。先前的研究指出,在登革熱病患血清中,具有抗血小板、抗內皮細胞與造成自體細胞傷害的自體抗體(autoantibody)存在。這研究指出在登革熱病毒感染的病程中,具有自體免疫反應(autoimmunity)的發生。相同的,在小鼠冠狀病毒感染的病程中,也偵測到具有自體抗體的產生。在本計劃中,我假設自體免疫反應所導致肺上皮細胞病理上的變化,可能是此疾病重要的病因之一。因此,本研究的目標就是去尋找在肺上皮細胞中,會受到SARS 自體抗體辨識的分子。首先,我利用二維凝膠電泳(two-dimensional gel electrophoresis)分別去分離人類肺上皮細胞(A549 cell line) 膜上與細胞質內不同之蛋白質。利用銀染和使用SARS病人血清進行免疫染色法,測定受到辨識的自體抗原(autoantigen)在二維凝膠電泳上斑點的位置,以及其所代表的等電點和分子量為何。我分析出此自體抗原的分子量為37kDa,而等電點大約在pH 6~8。接著將此受到免疫染色辨識的斑點,從二維凝膠電泳上的相對位置挖下,經由質譜儀去進行蛋白質胺基酸序列的鑑定。我們獲得五個候選的蛋白質,而其中分子量為37.5 kDa的甘油醛三磷酸去氫酶(glyceraldehyde-3-phosphate dehydrogenase 簡稱GAPDH)會受到SARS病人血清的辨識。此外,在A549細胞,以及已知會在感染嚴重呼吸道感染症候群後造成自體免疫傷害的其他器官,所分離出來的細胞株所具有的GAPDH,都會受到SARS自體抗體的辨識。最後,我們得知,抗GAPDH 免疫球蛋白 G (anti-GAPDH IgG) 會造成A549 cell死亡。本研究顯示出附著於膜上的GAPDH可能是引起SARS此疾病的自體抗原。而SARS病人血清中自體抗體與細胞膜上的GAPDH 之間的相互作用機制,可能是造成SARS病人表現多重器官功能衰竭症候群(multiple organ dysfunction syndrome)的主要病因之ㄧ。
英文摘要  Severe acute respiratory syndrome (SARS) is a type of viral pneumonia that is infected by atype of coronavirus. The lung is the major organ affected by SARS. It is postulated that viral infection induces severe damage of alveolar epithelial and capillary endothelial cells, leads to pulmonary, intra-alveolar organization and alveolar septal fibrosis. Eventually, SARS causes pulmonary fibrosis and atelectasis. Death may result from progressive respiratory failure due to alveolar damage. Previous report showed that anti-platelet and anti-endothelial cell autoantibodies were present in dangue patient serum. The involvement of autoimmunity in the pathogenesis of dengue virus infection was proposed. Similarly, the generation of auto-antibodies during mouse coronavirus infection has been demonstrated. Thus, in this proposal, autoimmune responses leading to the pathogenicity of lung epithelial cells is hypothesized as the clinical features of SARS disease. The aim of this study is to identify the specific molecules present in the epithelial cells that can be recognized by SARS auto-antibody. Human lung epithelial (A549) cell membrane and cytosol fractions were first resolved on 2D gels followed by silver stain and immunoblot to determine the 2D-spot, isoelectric point (pI) and molecular weight of the auto-antigens. A protein with a molecular weight of 37KDa and pI of pH 6~8 was recognized by the SARS patient serum. The immuno-positive spots were cut off and subjected to mass spectrometry for protein identification. Five potential candidates were obtained. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with a molecular weight of 37.5 KDa could be recognized by SARS patient serum. Furthermore, GAPDH was evident in A549 and other cell lines derived from different organs that may also damaged by autoimmune responses due to SARS. Finally, anti-GAPDH IgG was found to cause cell death of A549 cells. This study revealed that GAPDH was the potential SARS-induced autoantigen. The reaction between SARS patient serum autoantibody and membrane GAPDH may be responsible for the multiple organ dysfunction syndrome in SARS patients.
論文目次 中文摘要………………………………………………………………………i
英文摘要………………………………………………………………………iii
誌謝……………………………………………………………………………v
目錄……………………………………………………………………………vi
表目錄…………………………………………………………………………viii
圖目錄…………………………………………………………………………ix
文獻探討………………………………………………………………………1
第一節 嚴重急性呼吸系統症候群(SARS)簡介…………………………………………2
第二節 自體免疫反應(autoimmunity)……………………………………………………8
研究動機與目的………………………………………………………………12
實驗設計………………………………………………………………………14
材料與方法……………………………………………………………………17
實驗結果………………………………………………………………………41
第一節 確認A549 cell中SARS自體抗原的存在…………………………………... 42
第二節 了解此SARS自身抗原分子量與等電點的值………………………………..42
第三節 分析SARS 自體抗原的胺基酸序列………………………………………….43
第四節 進一步確認SARS 自體免疫抗原.....................................................................43
第五節 anti-GAPDH antibody 對A549 cell的影響......................................................46
第六節 受SARS感染之器官細胞是否都具有自身免疫抗體......................................46
第七節 確認A549細胞中GAPDH所在的位置……………………………………...47
討論……………………………………………………………………………48
結論……………………………………………………………………………51
結果圖表………………………………………………………………………52
參考文獻………………………………………………………………………67
自述……………………………………………………………………………71
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