臺灣博碩士論文加值系統

僵直性脊椎炎是一種緩慢進展的風濕性發炎疾病，它主要侵犯脊椎與關節結構並導致其功能損傷，患者通常有後背疼痛與脊椎僵硬等臨床特徵，並且嚴重影響罹病者的日常生活品質。過去的許多研究指出人類白血球抗原B27和僵直性脊椎炎有高度密切的相關性，並且大約有九成的僵直性脊椎炎患者可被檢測出HLA-B27呈陽性的狀況，但是其致病機轉尚未被清楚了解。
為了深入了解HLA-B27在僵直性脊椎炎中扮演的角色，實驗室學姐已經建立好兩株分別可利用doxycycline調控表現HLA-B2704（和僵直性脊椎炎有高度正相關的亞型）與HLA-B2706（和僵直性脊椎炎呈負相關的亞型）的HeLa細胞株。為了確保這些可誘導表現HLA-B27細胞株的純度，我首先利用流式細胞分選儀篩選出可誘導表現出綠色螢光的細胞，並且再透過西方墨點法與共軛焦顯微影像技術來確認進一步的確認，實驗結果發現HLA-B2704大部分表現在內質網中，而HLA-B2706則位於細胞膜的位置。另外我們也利用FRET技術觀察細胞內的HLA-B27是否會形成二聚體結構，由實驗結果發現HLA-B2704與HLA-B2706兩種亞型形成二聚體的能力似乎沒有太大的差別，這也意味著二聚體的形成似乎和AS疾病的產生沒有直接的關係。此外我們也利用FRAP技術追蹤HLA-B27分子在細胞流動的過程，發現HLA-B2704從ER流進Golgi的速度的確是較HLA-B2706緩慢。最後我們想要知道當細胞高度表現HLA-B2704後對小胞運輸所造的影響，利用二維電泳的方式比較誘導HLA-B2704表現前後細胞膜蛋白表現的差異，我們發現細胞過度表現HLA-B27前後G protein B-subunit在細胞表達的程度會有明顯的差異。由這個實驗指出HLA-B2704大量堆繼在ER後的確會影響細胞進行小胞運輸的效率，並且改變細胞膜上蛋白質的組成，接著導致細胞的傷害。

Ankylosing spondylitis (AS) is a chronic, progressive inflammatory rheumatic disease involving primarily the sacroiliac joints and the axial skeleton. The main clinical features are back pain and progressive stiffness of the spine, seriously reducing the quality of patient’s life. Previous researches have established that expression of human leukocyte antigen-B27 (HLA-B27) is strongly associated with ankylosing spondylitis. And approximately 90﹪of population carry the HLA-B27 allele, the pathologic role of HLA-B27 is still unclear.
In order to understand the role HLA-B27 plays in development of ankylosing spondylitis, we established two inducible HeLa cell lines that express EGFP fusion of either HLA-B2704 (AS associated subtype) or HLA-B2706 (AS non-associated subtype) upon addition of doxycycline. To ensure the purity of the inducible clones, EGFP-positive cells were further enriched by flow cytometric sorting. Inducible expression of EGFP-fusion HLA-B2704 and HLA-B2706 was further confirmed by immunoblotting and confocal imaging. Our data indicate that AS-associated HLA-B2704 is remained in the cytoplasm, colocalizing with ER, but AS non-associated HLA-B2706 is largely localized on the plasma membrane. Furthermore, the fluorescence resonance energy transfer (FRET) assay was performed using a spectrum-type CLSM to examine whether HLA-B27 is able to dimerize in cells. Surprisingly the result indicates that HLA-B2704 and HLA-B2706 displays similar ability in heavy-chain homodimer formation. Hence, our finding suggests that heavy-chain homodimers is unlikely directly related to pathogenesis of AS. In addition, we utilize the techniques of fluorescence recovery after photobleaching (FRAP) to track the movement HLA-B27 proteins in living cells, and the result shows that AS-associated HLA-B2704 is transported into Golgi apparatus at much slower rate than HLA-B2706. To investigate the effect of of HLA-B27 overexpression to vesicular transport, we isolated cell surface protein from non-induced and induced HLA-B27 cell lines, and the profiles of membrane proteins were analyzed by two dimensional electrophoretic analysis. The proteins exhibiting differential presence on the plasma membrane after induction were further identified by mass spectrometry. Through this approach we discovered that G protein β-subunit has differential presence level on the plasma membrane between non-induced and induced HLA-B27 cell lines. Hence, our data indicate that expression of HLA-B2704 potentially retard the overall efficiency of vesicular transport pathway, thereby altering the membrane associated protein composition and subsequently leading to dysfunction of the cells.

致謝 I
中文摘要 II
Abstract III
目錄 V
圖目錄 VIII
第一章、緒論 1
1-1 僵直性脊椎炎 1
1-2 人類白血球抗原-B27（Human Leukocyte Antigen-B27） 2
1-3 HLA-B27在僵直性脊椎炎中扮演的角色 4
第二章、實驗動機 7
第三章、材料與方法 8
3-1 材料 8
3-1.1 本研究中使用之藥品 8
3-1.2本研究中使用之試劑 11
3-1.3本研究中使用之Kit 12
3-1.4本研究中使用之酵素 12
3-1.5本研究中使用之抗體 13
3-1.6本研究中使用之緩衝溶液配方 13
3-1.7本研究中使用之SDS-PAGE 濃度及配方 17
3-1.8本研究中使用之細胞培養液 18
3-1.9本研究中使用之細菌培養液配方 18
3-1.10 本研究中使用的DNA質體 19
3-2 方法 20
3-2.1 小量質體DNA萃取 20
3-2.2 中量質體DNA萃取 20
3-2.3 細胞培養（cell culture） 21
3-2.4 細胞的轉染（Transfection） 22
3-2.5 西方墨點法(western blotting) 23
3-2.6 細胞萃取物製備（preparation of cell extract） 24
3-2.7去醣基化試驗（deglycosylation assay） 24
3-2.8 細胞螢光蛋白顯微影像分析 25
3-2.9 螢光能量共振轉移試驗 (Fluoresence resonance energy transfer assay) 26
3-2.10 Fluorescence Recovery After Photobleaching (FRAP) 26
3-2.11 細胞膜蛋白萃取 (Plasma membrane protein extraction) 26
3-2.12 二維電泳法（Two dimensional electrophoresis） 28
3-2.13 蛋白質濃度定量 30
3-2.14 銀染（Silver staining） 31
第四章、實驗結果 33
4-1 改善HeLa/TO HLA-B27 stable clone純度 33
4-2 HLA-B2706蛋白醣基化的現象 34
4-3 HLA-B27在細胞表現後形成二聚體的情況 34
4-4 追蹤HLA-B27在細胞流動的情況 35
4-5 過度表現HLA-B2704時對細胞膜表面蛋白質分布的影響 36
第五章、討論 37
參考文獻 40
附圖 43
自述 58

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