鼻咽癌的發生，與 Epstein-Barr virus (EBV) 的感染有高度相關性。EBV所編碼產生的第1 型潛伏性膜蛋白latent membrane protein 1 (LMP1) 會在受感染後的 latent stage II 和 III 時表現。LMP1已被報導和腫瘤生成有關，藉由活化nuclear factor-κB (NF-κB) 的路徑達成。在本研究中，將利用過度表現 LMP1 於鼻咽癌的細胞株 (TW04) 中，以研究在藥物處理 (Sodium Salicylate, Cisplatin and 5-Fluorouracil) 下，細胞增生、細胞凋亡、侵犯和轉移的能力。Sodium Salicylate 是一種非類固醇類發炎抑制劑 (NSAID) 能抑制 NF-κB 的下游路徑，例如 COX-1。Cisplatin 和 5-Fluorouracil 是傳統的化療用藥，用於多種癌症。我們的實驗結果顯示，在藥物處理下，LMP1 的過度表現會影響細胞增生、細胞凋亡、侵犯和轉移的能力。這個結果指出，在鼻咽癌的治療上 LMP1 的過度表現是個重要的指標。

Nasopharyngeal carcinomas (NPC) is highly induced by Epstein-Barr virus (EBV). EBV infection encoded latent membrane protein 1 (LMP1) is expressed in latent stage II and III of EBV infection nasopharyngeal cells. LMP1 was reported to be associated with increased tumorigenesis, through the activation of nuclear factor-κB (NF-κB). In this study, LMP1 overexpressing NPC cell line (TW04) was used for assays of cell proliferation, apoptosis and migration under drug (Sodium Salicylate, Cisplatin and 5-Fluorouracil) treatment. Sodium Salicylate is one of non-steroidal anti-inflammatory (NSAID) drug, which inhibits the downstream of NF-κB pathway, e.g., COX-1. Cisplatin and 5-Fluorouracil are traditional chemotherapy durgs used in many cancers. Our result shows that overexpression of LMP1 affects cell proliferation, apoptosis , invasion and migration ability. It indicates that LMP1-overexpression is an important marker for NPC therapy.

Contents

中文摘要 V
英文摘要 VI
英文縮寫表 VII
致謝 VIII

壹、 序言 1
1. 鼻咽癌 (Nasopharyngeal carcinomas, NPC) 1
(1) 鼻咽位置與解剖圖 1
(2) 病理特性與分期 2
(3) 化學治療 3
(4) 病理學及預後 4
2. Epstein-Barr virus 4
(1) 病毒特性 5
(2) 相關疾病 8
3. 第1型潛伏性膜蛋白 ( Latent membrane protein 1, LMP1 ) 8
(1) 生物功能 9
(2) 訊息傳遞 11
4. Sodium Salicylate, Cisplatin和5-Fluorouracil 12
(1) Sodium Salicylate 12
(2) Cisplatin 13
(3) 5-Fluorouracil 13

貳、 實驗目的 (Specific Aims) 15

參、 實驗流程 (Experiment Flowchart) 15

肆、 材料與方法 (Materials and Methods) 16
1. 實驗用藥 16
2. 細胞培養 16
(1) 材料 16
A、 細胞株
B、 藥品與試劑
(2) 培養方法 16
A、 培養液的配製
B、 解凍細胞
C、 繼代培養
D、 冷凍細胞
E、 細胞計數
3. 質體轉型 (Transformation) 18
4. 質體純化 (Purification) 與篩選 19
5. 菌株保存與冷凍 19
6. DNA濃度測量 20
7. 細胞轉殖 (Transfection) 20
8. MTT細胞存活率測試 20
9. 流式細胞儀分析 (Flow Cytometry Analysis) 21
10. 蛋白質萃取 21
11. 蛋白質濃度測量 22
12. 聚丙烯醯胺膠之配製(Sodium dodecyl sulfate Polyacrylamide Gel, SDS Gel) 22
13. 西方點墨法 (Western Blotting) 23
14. 細胞遷移分析 (Cell Migration Assay) 24
15. 細胞侵犯分析 (Cell Invasion Assay) 25
16. 軟瓊脂群落形成分析 (Soft Agar Colony Formation Assay) 25
17. 統計分析 (Statistical Analysis) 25

伍、 實驗結果 (Result) 27
1. 過度表現LMP1使鼻咽癌細胞株TW04快速增生 27
2. 過度表現LMP1在藥物處理下的存活能力 27
(1) Time-Dependent
(2) Dose-Dependent
3. 過度表現LMP1對細胞凋亡的影響 29
4. 過度表現LMP1對細胞遷移的影響 29
5. 過度表現LMP1對細胞週期的影響 30
6. 過度表現LMP1對細胞侵犯的影響 31
7. 過度表現LMP1對細胞群落形成的影響 31

陸、 實驗討論 (Discussion) 32
1. LMP1的過度表現使細胞增生速度加快 32
2. LMP1的過度表現對細胞存活能力的影響 32
3. LMP1的過度表現對細胞凋亡的影響 33
4. LMP1的過度表現對細胞遷移的影響 34
5. LMP1的過度表現對細胞週期的影響 35
6. LMP1的過度表現對細胞侵犯的影響 35
7. LMP1的過度表現對細胞群落形成的影響 35
8. 結論 36

柒、 圖表 (Figures and Tables) 37
Figure 1. 過度表現LMP1於TW04細胞株對細胞增生之影響 37
Figure 2. 在藥物處理下過度表現LMP1對細胞存活之時間影響 38
Figure 3. 在藥物處理下過度表現LMP1對細胞存活之劑量影響 39
Figure 4. 在藥物處理下過度表現LMP1對細胞凋亡之影響 42
Figure 5. 在藥物處理下過度表現LMP1對細胞遷移之影響 44
Figure 6. 在藥物處理下過度表現LMP1對細胞週期的影響 47
Figure 7. 過度表現LMP1對細胞侵犯的影響 49
Figure 8. 過度表現LMP1對細胞群落形成的影響 50
Table 1. 實驗結果統整 51
Figure 9. CDDP與SS對LMP1之作用機轉 52

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