臺灣博碩士論文加值系統

僵直性脊椎炎 (Ankylosing spondylitis，AS) 是一種緩慢進展的風濕性發炎疾病，此疾病會造成患者的脊椎結構受損，患者通常有後背疼痛與脊椎僵硬等臨床特徵。人類白血球組織抗原-B27 (Human Leukocyte Antigen-B27，HLA-B27) 與僵直性脊椎炎有的高度的相關性，90 ~ 95%的僵直性脊椎炎病人可以被檢測HLA-B27呈陽性的狀況。HLA-B27是主要組織相容性複合體I其中的分子，主要為免疫系統中負責抗原呈現的功能。但是目前對HLA-B27所引起的致病機轉尚未被了解清楚。HLA-B27重鏈蛋白質錯誤折疊和未折疊蛋白反應 (unfolded protein response，UPR) 調節的發炎反應與形成僵直性脊椎炎過程中扮演重要的角色。在此研究中，我們打算找出與僵直性脊椎炎中與僵直性脊椎炎正相關的HLA-B2704分子及與僵直性脊椎炎負相關的HLA-B2706分子間的差異。在HIG82(兔子關節滑膜細胞)中，與僵直性脊椎炎正相關的HLA-B2704表現在內質網，但當過度表現β2-microglobulin則會部分表現在細胞膜上，顯示說在單一表現HLA-B27時抗原呈現複合體組裝的過程中有缺失的情況。但在已表現β2m的狀況下加入胜肽，可以觀察到HLA-B2704小胞運輸僅有些微的改變。另外，我們也觀察在HIG82細胞中XBP1 messenger RNA (mRNA) 表現的情形，得知HLA-B27確實會活化UPR的產生。最後，在醣基化實驗中發現與僵直性脊椎炎正相關及負相關的HLA-B27分子皆有相同的醣化情形，推測與僵直性脊椎炎正相關的HLA-B2704並未運送到細胞膜，則可能錯誤運送到細胞的某處。因此，我們的結果指出與僵直性脊椎炎正相關的HLA-B2704可能無法正確運送到細胞膜結構上，而這樣的現象也許與AS產生有關聯。

Ankylosing spondylitis (AS) is a chronic and progressive inflammatory rheumatic disease that affects the axial skeleton, causing characteristic inflammatory back pain and structural and functional impairments. There is a strong correlation between the expression of Human leukocyte antigen B27 (HLA-B27) subtypes and occurrence of this disease. 90~95% of patients with ankylosing spondylitis carry specific HLA-B27 alleles. As a member of the MHC Class I molecules, the function of HLA-B27 is to present antigens to the immune system. However, how expression of specific HLA-B27 subtype induces inflammation is not entirely clear. There have been increasing interests in the critical role of protein misfolding and unfolded protein response (UPR) of the HLA-B27 heavy chain in modulation of the inflammatory response and progression of AS. In this study, we intend to identify the fundamental difference between AS associated and AS non-associated HLA-B27 molecules. In the rabbit synoviocyte cell line HIG82, AS associated HLA-B2704 protein appears to be trapped inside the endoplasmic reticulum (ER) and that co-expression of β2-microglobulin facilitates its export to the plasma membrane, suggesting a deficiency in the assembly process of the antigen-presenting complex. Addition of the arthritic peptides to HIG-82 showed little effect in assisting the export of HLA-B2704. Furthermore, spliced XBP-1 mRNA is present in HLA-B27 expressing HIG-82, indicating that UPR is activated. On the other hand, although glycosylation status appears to be similar for both AS associated and AS non-associated HLA-B27 molecules, AS associated HLA-B2704 is not presented to the plasma membrane, giving rise to the possibility that AS associated HLA-B27 is targeted to incorrect location inside the cells. Thus, our data indicates that the AS associated HLA-B27 molecules is possibly transported to incorrect target membrane structures, suggesting a potentially putative disease-causing molecular mechanism.

謝誌 I
摘要 II
Abstract IV
目錄 VI
圖目錄 IX
第一章、序論 1
1-1 僵直性脊椎炎 1
1-2 人類白血球抗原 2
1-2.1 人類白血球抗原 (Human Leukocyte Antigen， HLA) 2
1-2.2 人類白血球抗原-B27 (Human Leukocyte Antigen-B27，HLA-B27) 4
1-3 僵直性脊椎炎與HLA-B27之關係 5
第二章、實驗動機 9
第三章、材料與方法 10
3-1 材料 10
3-1.1 抗體 10
3-1.2 溶液配方 11
3-1.3 SDS-PAGE濃度配方 13
3-1.4 細胞培養液 14
3-1.5 細菌培養液 15
3-1.6 引子 17
3-2 方法 19
3-2.1 質體的構築 (plasmid construction) 19
3-2.2 電穿孔質體的轉型作用 (transformation) 22
3-2.3 質體萃取 (plasmid extraction) 22
3-2.4 RNA萃取 (purication of RNA) 24
3-2.5 反轉錄聚合酶鏈鎖反應 (reverse transcription-PCR) 25
3-2.6 即時定量聚合酶連鎖反應 (real-time PCR) 25
3-2.7 西方墨點法 (western blotting) 26
3-2.8 細胞轉染 (transfection) 26
3-2.9 免疫螢光染色 (immunofluorescence) 27
3-2.10 細胞萃取 (cell extraction) 28
3-2.11 去醣基化試驗 (deglycosylation assay) 28
3-2.12 蛋白質定量 (protein quantitative analysis) 29
3-2.13 細胞膜蛋白萃取 (plasma membrane protein extraction) 30
第四章、實驗結果 32
4-1 建立HeLa/TO-HLA-B27 stable clone 32
4-2 HLA-B27-EGFP於HeLa細胞中的分佈情形 32
4-3 HLA-B27於HeLa細胞中蛋白醣基化的情形 33
4-4 HLA-B27-EGFP及過度表現β2m於HIG82細胞中的分佈情形 34
4-5 HLA-B27於HIG82細胞中蛋白醣基化的情形 35
4-6 HIG82中表現HLA-B27及過度表現β2m時對細胞蛋白質分佈的影響 36
4-7 於不同細胞中觀察表現HLA-B27及過度表現β2m時誘導ER stress的情形 37
4-8 HLA-B27-EGFP於HIG82細胞中lysosome分佈的情形 37
4-9 HLA-B27於HIG82細胞中細胞激素mRNA表現的情形 38
第五章、討論 39
參考文獻 43
附圖 46

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