目前在人體中發現有四種SUMO的存在，分別為SUMO-1, -2, -3和-4。其中大多數的SUMO-1/2是位於細胞核中，但SUMO-1與SUMO-2蛋白的同質性只有44%。已知可被SUMO-1修飾的目標蛋白已超過50幾種，包含了轉錄因子與共同轉錄因子、調控因子、核體蛋白、核孔複合蛋白、DNA修復相關的蛋白與一些病毒蛋白。然而對於SUMO-2修飾的目標蛋白目前所知的不多，可能與環境壓力有關。藉由酵母菌二次雜交的方法中發現，SUMO-1可與Fas/APO-1和TNF受體連結，但此是否對於生物性壓力所造成的細胞凋亡有關尚未清楚。幽門螺旋桿菌為一已知的胃癌致病因子，所以我們利用幽門螺旋桿菌可引起細胞的凋亡，作為一種生物性的壓力，觀察SUMO-1/2在細胞受到感染下對於細胞凋亡的影響。當過度表現SUMO-1/2在細胞中，12小時後並不會影響細胞的凋亡；但若受幽門螺旋桿菌感染後，SUMO-1/2則會加強細胞凋亡的產生。此外，當細胞過度表現SUMO-1/2在48小時後會增加細胞的凋亡產生，然而受幽門螺旋桿菌感染的細胞群中，只有SUMO-2會明顯的增加細胞的凋亡。所以在受到幽門螺旋桿菌感染的細胞中，SUMO-2相較於SUMO-1更能增強細胞凋亡的產生。不活化的SUMO-1/2，即SUMO-1/2失去相撲化(sumoylation)能力則使細胞凋亡影響減弱，顯示SUMO的相撲化與細胞的凋亡有關。另外，藉由幽門螺旋桿菌感染2小時會使SUMO-2出現在細胞質的細胞比例上升22%，而SUMO-1則為11%。Leptomycin B可抑制SUMO-1的出核，但對於SUMO-2則無影響。Leptomycin B也抑制了SUMO-1在受感染的細胞中所增加的細胞凋亡，但依然無法抑制SUMO-2。因此，本研究認為SUMO-1/2在細胞受到幽門螺旋桿菌感染短時間中會由細胞核移至細胞質中而加強細胞凋亡的產生。其中SUMO-1的出核受到出核因子的調控，而SUMO-2則否。

Four small ubiquitin-like modifier (SUMO) isoforms termed SUMO-1, -2, -3 and -4 have been identified in human. Most SUMO-1/2 proteins are localized in nucleus, whereas SUMO-1 protein exhibits 44% homolog with SUMO-2 protein. Over 50 proteins have been identified as the target proteins for SUMO-1 modification and these include transcription factors, their cofactors, regulators, nuclear body proteins, nuclear pore complex proteins, DNA repair proteins, and viral proteins. However, only a handful of SUMO-2 targets are known and SUMO-2 modification may response to environmental stress. SUMO-1 may interact with Fas/APO-1 and TNF receptor 1 on yeast two hybrid interactions; however, it is not clear whether SUMO would enhance apoptosis or response to biological stress. Helicobacter pylori (H. pylori) defined as a gastric carcinogen is definite a biological stress to the cells. It causes gastric epithelial cell damage by apoptosis. In this study whether the SUMO-1/2 pathway constitutes an element of the cellular response to the H. pylori infection was examined. Overexpression of SUMO-1/2 for 12 hours had no effects on the apoptotic activities of cells; however it enhanced apoptosis during H. pylori infection. Overexpression of SUMO-1/2 for 48 hours increased the apoptosis of cells; however only SUMO-2 enhanced apoptosis significantly during H. pylori infection. The enhancements are more powerful for SUMO-2 than that of SUMO-1. Inactive SUMO, a cytoplasm dispersed sumoylation-incompetent mutant, eliminates such activities, suggesting that sumoylation or SUMO interactions are involved in the apoptotic enhancement. The percentages of cells with cytoplasmic SUMO-2 were increased 22% by H. pylori infection for 2 hours and SUMO-1 were increased 11%. The translocalization of SUMO-1 was blocked by leptomycin B; however, it did not work on SUMO-2. Leptomycin B could also inhibit SUMO-1 enhanced apoptosis during H. pylori infection, whereas it had no effects on SUMO-2. It is concluded that SUMO-1/2 pathway constitutes an element of the cellular response to H. pylori infection by enhancing apoptosis through shuttling from nucleus to cytoplasm. SUMO-1 is via a CRM1-dependent pathway while SUMO-2 is via a CRM1-independent pathway.

中文摘要……………………………………………………………… I
英文摘要……………………………………………………………… II
縮寫表………………………………………………………………… III
壹、緒論……………………………………………………………… 1
1. 幽門螺旋桿菌 (Helicobacter pylori)………………………….. 1
1-1. 尿素酶(Urease)之作用…………………………………….. 2
1-2. Cag Pathogenicity Island (cag PAI)之毒性………………… 4
1-3. Vacuolating Cytotoxin A (VagA)之毒性………………......... 6
1-4. 幽門螺旋桿菌與細胞凋亡的關係……………………….... 9
2. SUMO…………………………………………………………… 10
2-1. SUMO-1與泛激素(Ubiquitin).……………………………. 10
2-2. 相撲化(Sumoylation)……………………………………… 12
2-3. SUMO的同質體(Isoform)………………………................ 14
2-4. SUMO-1在細胞中的位置…………………………………. 15
2-5. SUMO-1在細胞中的功能………………………………….. 16
2-5-1. 調控蛋白質在細胞中的位置………………………... 16
2-5-2. 轉錄的因子的調控…………………………………. 17
2-5-3. 穩定蛋白質的作用………………………………….. 19
2-5-4. 核體的組成………………………………………….. 20
2-6. SUMO-1與細胞凋亡的關係………………………………. 21
2-7. SUMO-2/3在細胞中的位置……………………………….. 22
2-8. SUMO-2/3在細胞中的功能………………………………... 22
3. 實驗室之前的研究…………………………………………….. 23
貳、 研究目的………………………………………………………… 25
參、 實驗材料及方法………………………………………………… 26
1. 細胞培養………………………………………………………… 26
1-1. 細胞株……………………………………………………… 26
1-2. 細胞培養液的配置………………………………………… 26
1-3. 繼代培養…………………………………………………… 28
1-4. 冷凍保存…………………………………………………… 29
1-5. 解凍………………………………………………………… 30
2. 轉植 (Transfection)…………………………………………….. 30
2-1. 轉植質體 (plasmids)……………………………………… 30
2-2. 細胞計數…………………………………………………… 31
2-3. 細胞種植 (seeding)………………………………………... 31
2-4. 細胞轉植…………………………………………………… 32
2-4-1. Superfect轉植質體…………………………………….. 32
2-4-2. Lipofectamine 2000轉植質體…………………………. 33
3. 感染 (Infection)…………………………………………………. 34
3-1. 細菌………………………………………………………… 34
3-2. 細菌培養…………………………………………………… 34
3-3. 菌量計數…………………………………………………… 35
3-3-1. 比色管計數…………………………………………… 35
3-3-2. OD600吸光值計數…………………………………….. 35
3-4. 細胞感染…………………………………………………… 36
4. 螢光顯微鏡分析 (Fluorescence microscope analysis)………… 36
4-1. Annexin-V 染色…………………………………………… 36
4-2. DAPI染色………………………………………………….. 37
5. 流式細胞技術(Flow Cytometry)……………………………….. 39
5-1. Annexin-V assay……………………………………………. 39
5-2. Flow Check…………………………………………………. 40
5-3. Sample anaylsis…………………………………………….. 41
5-4. Close………………………………………………………… 42
肆、結果……………………………………………………………….. 43
1. SUMO-1/2的表現對幽門螺旋桿菌所引起細胞凋亡的影響…. 43
2. 不活化的SUMO-1/2對幽門螺旋桿菌所引起細胞凋亡的影響 46
3. SUMO-1/2在受到幽門螺旋桿菌感染後的細胞中位置的變化.. 48
3-1. SUMO-1/2在受到幽門螺旋桿菌感染不同時間後對於在細胞中位置的改變………………………………………...
51
3-2. SUMO-1/2在受到不同濃度幽門螺旋桿菌感染後對於在細胞中位置的改變………………………………………...
53
4. Leptomycin B對於細胞中SUMO-1/2出核的影響……………. 55
5. SUMO-1/2在細胞中位置的改變對細胞凋亡的影響…………. 57
伍、 討論………………………………………………………………. 61
1. SUMO與細胞凋亡的關係………………………………………. 61
2. SUMO是藉由相撲化(Sumoylation)影響細胞的凋亡………….. 62
3. 幽門螺旋桿菌會引起AGS細胞凋亡的產生…………………... 63
4. 幽門螺旋桿菌感染後SUMO-1/2對細胞凋亡的影響…………. 63
5. 幽門螺旋桿菌感染對SUMO-1/2在細胞中分佈的影響……… 65
6. SUMO-1/2在細胞中的分布對細胞凋亡的影響……………….. 68
7. 總結……………………………………………………………… 70
8. 未來工作………………………………………………………… 71
陸、參考文獻…………………………………………………………… 72
柒、附錄………………………………………………………………… 77
附錄一 SUMO所引起細胞凋亡比例的原始資料……………….. 77
附錄二 SUMO在細胞中分佈的差異與凋亡比例影響的原始資料…………………………………………………………..
83
附錄三 pDsRed1-C1轉植載體…………………………………… 91
附錄四 SUMO-1/2的胺基酸序列………………………………… 92

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