許多的細胞外刺激皆會引起 MAPK 途徑的活化，這些刺激可能為紫外光、放射線、熱休克或促發炎細胞激素等。其中 MAPK 的活化主要藉上游激酶磷酸化 MAPK 活化環中的 TXY 區域。MAPK 蛋白被認為在調控細胞凋亡上扮演重要的角色；它也被認為和敗血症及關節炎造成的原因有密切的關係。而蛋白質轉譯後的相撲化修飾作用 (SUMOylation) 也負責調控許多細胞功能，諸如訊息傳遞、蛋白質的標靶運送、穩定蛋白質、轉錄作用的活化或調控細胞凋亡等。先前本實驗室研究結果得知當幽門螺旋桿菌感染時，SUMO-1的 mRNA 及蛋白質會表現較多，MAPK 途徑有被活化，細胞凋亡現象也有上升的情形。因此我們推測幽門螺旋桿菌感染相關的細胞凋亡現象可能與相撲化修飾作用及 MAPK 途徑有關係。
本實驗研究結果顯示當胃癌上皮細胞大量表現 RFP-SUMOs 或GFP-MAPK 時，MAPK 的磷酸化及細胞凋亡有上升的趨勢；特別在幽門螺旋桿菌感染時更加顯著。而此現象可被 MAPK 抑制劑所抑制。當 AGS 細胞大量表現GFP-MAPK 時，MAPK蛋白質磷酸化及細胞凋亡也有上升的趨勢，因此我們推測 MAPK 蛋白為 SUMOs 的修飾蛋白。

Diverse extracellular stimuli, including ultraviolet light, irradiation, heat shock, proinflammatory cytokines, trigger activation of MAPK pathway through phosphorylation on a TGY motif within the kinase activation loop. Protein MAPK appears to play a major role in apoptosis. It has been causally implicated in sepsis and arthritis. The translational small ubiquitin related modifier (SUMO) modification of proteins has been shown to play multiple functional roles in several cellular processes, including signal transduction, protein targeting, stabilization, transcriptional activation and apoptosis. Our previous study demonstrated that the expression levels of SUMO-1 rnRNA and proteins were enhanced in Helicobacter pylori infected human gastric epithelial cells. The activation of MAPK pathway and cellular apoptosis of AGS cell lines were increased during Helicobacter pylori infection. It was hypothesized that Helicobacter pylori functioning as a biological stress that induced MAPK mediated apoptosis which may be regulated by sumoylation.
Results showed that MAPK phosphorylation and cellular apoptosis were enhanced in RFP-SUMOs or GFP-MAPK expressing cells, especially during Helicobacter pylori infection. It was inhibited by pretreatment of MAPK inhibitor. The enhanced phosphorylation and apoptosis were observed during GFP-MAPK overexpression. It’s suggested that MAPK is a target protein for SUMOs.

中文摘要……………………………………………………………… 1
英文摘要……………………………………………………………… 2
縮寫表………………………………………………………………… 3
壹、緒論……………………………………………………………… 6
一、幽門螺旋桿菌 (Helicobacter pylori)…………………………… 6
1.1細胞毒素相關基因致病性基因島嶼
(Cytotoxin associated gene pathogenicity island, Cag-PAI)… 7
1.2 細胞毒素相關基因A (Cytotoxin associated gene A, CagA). 9
1.3 液泡式細胞毒素A (Vacuolating cytotoxin A, VacA)……… 11
1.4 幽門螺旋桿菌與細胞凋亡的關係………………………… 12
二、絲裂原活化蛋白激酶途徑 (MAPK pathway)………………… 14
2.1 p38 MAPK.………………………………………………… 15
2.2 p38 MAPK活化途徑及其功能…………………………… 16
2.3 p38α 結構與功能…………………………………………… 19
2.4 p38 MAPK於細胞內之位置與其功能…………………… 20
2.5 p38 MAPK與細胞凋亡的關係…………………………… 21
三、相撲蛋白 (Small ubiquitin-related modifiers, SUMOs)……….. 22
3.1 相撲化 (SUMOylation) …………………………………… 23
3.2 相撲蛋白在細胞中的功能………………………………….25
3.2.1 調控核體 (nuclear bodies) 的組成………………………25
3.2.2 調控蛋白質在細胞中的位置……………………………..26
3.2.3 調控蛋白質的活性及穩定性…………………………….. 28
3.3 相撲蛋白與細胞凋亡的關係………………………………. 30
貳、研究目的………………………………………………………… 31
參、實驗材料及方法………………………………………………… 32
一、 DNA Cloning…………………………………………………… 32
二、 E. Coli transformation及重組基因之確認……………………. 36
三、由點導引的突變產生法 (Site-directed mutagenesis) …………. 39
四、細胞培養…….………………………………………………… 41
五、基因轉殖……………………………………………………… 45
六、幽門螺旋桿菌感染 (Hp infection) …………………………… 47
七、蛋白質電泳與西方墨點法 (Western blots, WB) ……………… 49
八、螢光顯微鏡分析 (Fluorescence microscope analysis) ………… 55
九、免疫細胞化學法 (Immunocytochemistry, ICC) ……………… 57
十、細胞週期測試 (Cell cycle assay) .……………………………… 60
十一、蛋白質表現、純化及保存…………………………………… 62
十二、試管內相撲化反應 (In vitro sumoylation assay) …………… 67
肆、結果……………………………………………………………… 69
一、過度表現SUMOs時可放大p38的活化……………………… 69
二、 SUMOs可與內生性p-p38形成核體結構…………………… 72
三、野生型p38-wt及其突變質體的建構…………………………… 77
四、p38-79*與p38-P*不具放大細胞凋亡之功能…………………… 80
五、p38-79*可被磷酸化的能力明顯的下降………………………… 83
六、SB203580 抑制含 GFP-p38-wt 細胞凋亡之功能…………… 86
七、p38-wt，p38-79* 及 p38-P* 與 SUMO形成核體…………… 89
八、p38 聚集於 SUMOs-PML 核體上…………………………… 93
九、p38的試管中相撲化反應 (in vitro sumoylation) ……………… 96
伍、討論……………………………………………………………… 101
陸、未來工作………………………………………………………… 109
柒、參考文獻………………………………………………………… 110
捌、附錄……………………………………………………………… 127
一、SUMOs誘發p38 MAPK磷酸化的原始資料…………………… 127
二、GFP-p38-wt、GFP-p38-79*、GFP-p38-P*影響細胞凋亡的原始
資料……………………………………………………………… 128
三、GFP-p38-79*對於其磷酸化修飾功能之影響的原始資料……… 129
四、SB203580影響GFP-p38誘導之細胞凋亡的原始資料……… 130
圖目錄
圖一、p38 的活化在含 RFP-SUMOs 的細胞中明顯的增加……… 70
圖二 (A, B)、H. pylori 感染時內生性的p-p38在細胞中表現的情形 73
圖二 (C, D)、H. pylori 感染時，內生性的p-p38與不活化的
RFP-SUMOΔs 在細胞中作用的情形………………………… 75
圖二 (E, F)、H. pylori 感染時，內生性的p-p38與活化的RFP-SUMOs在細胞中作用的情形…………………………… 76
圖三 (A, B)、野生型GFP-p38及其突變質體的建構……………… 78
圖三 (C, D)、野生型GFP-p38及其突變質體蛋白的表現………… 79
圖四、GFP-p38-P* 與 GFP-p38-79* 無法放大細胞凋亡之功能… 81
圖五、突變型的GFP-p38-79* 可被磷酸化的能力明顯的下降…… 84
圖六、SB203580 抑制含 GFP-p38-wt細胞凋亡之功能…………… 87
圖七、GFP-p38 與 RFP-SUMOs 在細胞中的交互作用…………… 90
圖八、GFP-p38-wt和其突變蛋白與 RFP-SUMOs-PML共存於核體 94
圖九、p38 的試管中相撲化反應 (A) …….………………………… 98
圖九、p38 的試管中相撲化反應 (B) …….………………………… 99
αΔ

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