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研究生： 陳靜儀
Chen, Jing-Yi
論文名稱： 人類遺傳疾病中白血球分子的表現與調控機制 研究
Molecular Mechanisms of Abnormal Expression of Leukocyte Molecules in Human inherited Diseases
指導教授： 謝奇璋博士
Shieh, Chi-Chang
學位類別： 碩士
Master
系所名稱： 醫學院 - 微生物及免疫學研究所
Department of Microbiology & Immunology
論文出版年： 2002
畢業學年度： 90
語文別： 中文
論文頁數： 68
中文關鍵詞： 唐氏症 、黏著分子 、慢性肉芽腫病 、呼吸爆發 、NADPH氧化脢 、氧活性物質
外文關鍵詞： ROS, gp91, NADPH oxidase, respiratory burst, LFA-1, Down syndrome, chronic granulomatous disease
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    • 中文摘要

    免疫系統的防禦機制常藉由許多蛋白質例如黏著分子、細胞激素、趨化物質、白血球酵素和白血球表面與抗原結合的接受器的參與才能順利執行。如果這些蛋白質發生錯誤，免疫系統將無法發揮其防禦的功能而造成免疫缺陷疾病。遺傳疾病是因為染色體DNA的改變所造成之疾病。基因上的改變常常對免疫反應有著直接或間接的影響，也因此這些遺傳疾病病患會產生異常的免疫反應。我們藉著分析遺傳疾病中基因改變所造成的免疫缺陷，去了解基因在免疫反應的分子機轉所扮演的角色。
    唐氏症病人出生時即易受到感染。唐氏症是由於第21對染色體異常所造成的遺傳性疾病。以往的研究報告指出，唐氏症病人有免疫缺陷的病徵產生；除此之外，還有研究指出淋巴細胞表面的LFA-1的表現有增加，這是由於LFA-1的β鏈蛋白(CD18)的基因是位於第21條染色體上，因此在唐氏症病患細胞上有增加的現象。LFA-1對於胸腺細胞在發育成熟過程中傳遞一個重要的訊息，所以在唐氏症病人中是否因過度表現的LFA-1造成不同淋巴細胞分群在發育過程中的異常，進而引起唐氏症病人體內的免疫缺陷。一些研究調查發現這些不同淋巴細胞分群的比例改變在胎兒時期便開始。根據此我們想要知道LFA-1的表現是否隨著年齡有增加的現象。此外，我們也想瞭解不同分群的淋巴細胞上LFA-1的表現是否不同。我們發現在CD8+淋巴細胞及CD45RO+淋巴細胞在唐氏症病童中有較高的表現比例但在CD4+淋巴細胞則沒有。另外我們發現CD4+淋巴細胞、CD8+淋巴細胞及CD45RO+淋巴細胞上的LFA-1表現隨著年紀增加並無明顯的增加。我們推測LFA-1的表現在不同分群的淋巴細胞的發育成熟扮演重要角色。
    慢性肉芽腫病(CGD)是一種遺傳性的免疫缺陷疾病。由於白血球表面的NADPH oxidase的缺陷，使得白血球不能夠產生殺死外來病原的氧活性物質(reactive oxygen species，ROS)。NADPH oxidase由五個次單位組成:p40phox、p47phox、p67phox、p22phox和gp91phox。這五個部分的前三個是位於細胞質中，另兩個部分則分布在細胞膜上形成叫作flavocytochrome b558。大多數慢性肉芽腫病人為gp91phox的突變所造成，由於調控gp91phox的基因是位於性染色體上，所以此型的病人我們稱之為X-linked (X91) CGD。為了瞭解CGD的致病中modification對蛋白質合成的監控系統所扮演的角色，我們分析了的 X-linked CGD病人中gp91phox的突變的表現情形為何。在CGD病人中,在gp91phox的點突變造成第338個胺基酸histidine變為tyrosine。我們觀察CGD病人細胞表面的gp91phox表現發現表現在細胞質的gp91phox並未表現在細胞表面。進一步分析細胞質中gp91phox的表現分佈，我們發現gp91phox滯留在內質網中無法送至細胞膜上表現；在與calnexin結合的分析中可知，滯留在內質網中的gp91phox是由於與calnexin結合以致於無法送出內質網到細胞膜表現。因此，我們推測由於gp91phox上的點突變使得gp91phox無法正確褶疊而與calnexin結合滯留在內質網中。這些結果表明modification對蛋白質合成的監控系統在X-linked CGD的致病因中扮演重要的角色。

    Abstract

    Immune system plays a very important role in preventing host from being infected by pathogenic microorganisms. Many proteins such as cytokines, adhesion molecules, and chemoattractants participate in immune responses. Inherited diseases are caused by changes in chromosomal DNA. The alternations of genes may directly or indirectly affect immune responses and result in immunodeficiency in patients with inherited diseases. In this study, we investigated the immunodeficiencies in inherited diseases to understand how the alterations in genes affect the molecular mechanisms involved in immune responses.
    Individuals with Down syndrome are at high risk of infection. Down syndrome (DS) is a disease caused by trisomy of chromosome 21. Previous studies suggested that the immunodeficiency in patients with Down syndrome results from the overexpression of CD18, which is encoded on chromosome 21. Lymphocyte function associated antigen (LFA-1), which is composed of α chain (CD11a) and β chain (CD18, β2 integrin), plays an important role in the development of lymphocyte in thymus and enhancement of signal transduced from T cell receptor. The overexpression of LFA-1 was believed to lead to the abnormal development and function of lymphocytes in patients with DS. We analyzed LFA expression in lymphocyte subpopulations in DS children and in age matched controls. Although older children without DS tend to increase their expression of lymphocyte LFA-1 when compared with younger normal children, DS patients showed no age-associated increase in lymphocyte LFA-1 expression. Two-color analysis with CD4/CD8 and LFA-1 in patients and control showed that proportions of CD4+ lymphocytes were comparable in DS patients and controls, while the proportions of CD8+ lymphocytes in younger DS patients were higher when compared with age-matched controls and close to the proportion in the older DS groups. Proportions of memory lymphocytes expressing the CD45RO isoform were higher in both younger and older DS patients when compared with age-matched control groups. The LFA-1 expression levels on CD45RO+ lymphocytes from younger DS patients were higher than the levels of the controls and declined in the older DS group.
    Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease. Because of the deficiency of leukocyte NADPH oxidase, leukocyte cannot produce reactive oxidants to kill invading microorganisms. Most of CGD patients have mutations in the membrane component gp91phox and are inherited as X-linked (X91) CGD. In order to elucidate the role of posttranslational molecular quality control machinery in pathogenesis of CGD, we analyzed the effects of CGD mutation of on the characteristics of an X-linked CGD patient with a point mutation that causes a single amino acid change in the predicted FAD-binding site of gp91phox. The production of gp91phox could be detected in ER, but the expression on cell surface was not detectable. Calnexin binding appeared to be important for ER retention of these mutant gp91phox. These results indicate that the posttranslational molecular quality control machinery in endoplasmic reticulum (ER) plays an important role in pathogenesis of X-linked CGD.

    目錄 英文摘要 1 中文摘要 3 緒論 5 第一部分：唐氏症病患體內不同次群體的淋巴細胞上LFA-1表現並無隨年齡增長而增加之趨勢 7 實驗材料與方法 10 A. 檢體來源 10 B.週邊單核血球細胞的純化 10 C.流體細胞螢光分析儀測定細胞表面CD4、CD8、CD45RO及LFA-1之表現 12 實驗結果 14 討論 18 第二部分：細胞內轉譯後分子監控機制造成白血球NADPH oxidase的缺陷引起慢性肉芽腫病的發生 20 實驗材料與方法 24 A. Case report 24 B. CGD病人之gp91phox的表現型 24 C. EBV-transformed B cell line之建立 25 C.1. Epstein-Barr virus (EBV) solution的製備 25 C.2. 週邊單核細胞 (Peripheral Blood Mononuclear Cells, PBMC)的純化 26 C.3. EBV-transformed B cell line之建立 26 D. 細胞膜上gp91phox的表現 27 D.1. Cell lysate的製備 28 D.2. 免疫沉澱法(Immunoprecipitation) 30 D.3. SDS- PAGE電泳分析 32 D.4. 西方墨點法(Western blotting) 34 E. Metabolic assay 36 E.1. Cell lysate的製備 37 E.2. 免疫沉澱法(Immunoprecipitation assay) 38 E.3. Glycosidase assay 39 E.4. SDS- PAGE電泳分析 39 F. 免疫螢光細胞染色(Immunofluorescence staining assay) 40 G. 共軛焦顯微鏡(Confocal microscope) 42 實驗結果 43 討論 46 References list 48 附表及附圖 53 圖表目錄 Fig.1 在唐氏症病童及健康小孩體內淋巴細胞上LFA-1的表現情形 53 Fig.2 唐氏症與健康小孩體內CD4+和CD8+淋巴細胞的表現比例 54 Fig.3 唐氏症與健康小孩體內CD4+和CD8+淋巴細胞表面LFA-1的表現分析 55 Fig.4 唐氏症與健康小孩體內CD45RO+淋巴細胞表現分析 56 Fig.5 唐氏症與健康小孩體內CD45RO+淋巴細胞表面LFA-1的表現分析 57 Fig.6 分析CGD病人與Normal control EBV-transformed cells表面gp91phox的表現 58 Fig.7 利用Pulse-chase metabolic labeling and glycosidase analysis分析CGD病人與Normal control EBV-transformed cells其gp91phox在內質網中形成情形 59 Fig.8 以Pulse-chase metabolic labeling and coprecipitation assay分析calnexin 與gp91phox的結合情形 60 Fig.9共軛焦顯微鏡分析CGD病人與Normal control EBV-transformed cells中gp91phox的表現分佈 61 Appendix I. 文獻中報告的唐氏症病人免疫功能缺失 62 Appendix II. 慢性肉芽腫病依基因突變位置的分類 63 Appendix III “calnexin循環” 64 Appendix IV ROS的生成 65 Appendix V 活化的吞噬細胞NADPH oxidase各次單位的組合情形 66 Appendix VI 三種不同基因位置突變的X-linked gp91phox CGD病人 67 Appendix VII Lack of age-associated LFA-1 up-regulation and impaired ICAM-1 binding in lymphocytes from patients with Down syndrome 68 Appendix VIII Molecular quality control machinery contributes to the leukocyte NADPH oxidase deficiency in chronic granulomatous disease 69

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