臺灣博碩士論文加值系統

口腔癌 ( OSCC ) 屬於頭頸癌 ( HNSCC ) 的一種，相較往年的統計資料，口腔癌的致死率有逐年升高的趨勢。 Tid1屬 DNAJ protein 的家族成員能和 Heat shock protein ( HSP70 ) 交互作用，在口腔癌中扮演腫瘤抑制者 ( tumor suppressor ) 的角色，且能促進對致癌蛋白( oncoprotein ) 的降解 ( degradation )。 Tid1有兩種 isoforms ， Tid1 long form ( Tid1-L ) 和 Tid1 short form ( Tid1-S )。兩種 isoforms 在細胞中擁有不同功用。實驗室先前利用蛋白質體學方法 ( proteomics ) 尋找和 Tid1結合的蛋白質，首先以定點突變法 ( site-directed mutagenesis ) 將 Tid1和 HSP70結合的 DnaJ domain HPD loop 突變，進而以親和層析法純化Tid1蛋白複合物，再以質譜儀鑑定且相互比較，發現Tid1-SMu會和BAG2蛋白特異性結合，但BAG2在口腔癌中所扮演的角色仍然還不清楚。
本論文首先以免疫共沉澱和共軛焦顯微鏡，確認在細胞內 Tid1確實和 BAG2有結合，並且兩者可共同存在於粒線體中。對於在口腔癌細胞中過度表現或抑制 BAG2表現，皆對癌細胞表型有一定之影響。因此統整本論文之結果發現 Tid1和 BAG2在口腔癌細胞中具有一定影響力。

Oral squamous cell carcinoma (OSCC) is a major subtype of head and neck squamous cell carcinoma (HNSCC). Depanding on the data, OSCC mortality rate is increasing year by year. Tid1, a DnaJ cochaperon protein, can bind with HSP70, functions as a tumour suppressor in OSCC tumourigenesis. We previously used systemic proteomics analysis to identify Tid1 interacting protein. The HPD loop of DnaJ domain could bind with HSP70 which on Tid1 was mutated by site-directed mutagenesis. Through affinity chromatography and mass spectrometry we identified BAG2 that specifically interact with Tid1 short form (Tid1-S) lacking the functional DnaJ domain. However, the role of BAG2 in OSCC tumourigenesis remains unclear.
First, co-immunoprecipitation analysis and confocal microscope were performed to confirm the protein interaction between BAG2 and Tid1. Besides, BAG2 and Tid1 are colocolization in mitochondrial. No matter overexpression or silencing BAG2 in OSCC cells, it always has effects on their phenotypes. Together, these findings suggest that, Tid1 and BAG2 have important roles in OSCC cells.

目錄
致謝 i
中文摘要 ii
Abstract iii
目錄...................................................................................................iv
圖目錄 ix
附錄 xi
壹、緒論 1
一、口腔癌 1
二、 Tid1背景研究之介紹 2
2.1源起 2
2.2 Drosophila tumor suppressor Tid56 2
2.3 Tid1的基因結構 (genomic structure) 3
2.4 Tid1 及其結合之受質蛋白質 4
2.5 Tid1和腫瘤生成 ( tumorigenesis ) 的關係 6
2.6 Tid1 與Heat shock protein 70 ( HSP70 ) 9
三、 以蛋白質體學研究 Tid1 結合之受質蛋白 11
3.1以 site-directed mutagenesis 找尋 Tid1 之受質蛋白 11
3.2找尋標的蛋白質的方法 12
3.3 分析標的蛋白質結果 12
四、BAG2之介紹 13
4-1. BAG family 13
4-2. BAG2簡介 14
貳、研究目標 16
參、材料與方法 17
一、實驗材料 17
二、實驗方法 26
2-1. 聚和酶連鎖反應 ( polymerase chain reaction，PCR ) 26
2-2. PCR cloning 26
2-3. DNA洋菜膠體 ( agarose gel ) 電泳 27
2-4. 大腸桿菌轉形作用 ( Heat-shock transformation ) 27
2-5. 細菌的培養 28
2-6. PCR菌落快速篩選法 ( PCR quick screening ) 29
2-7. 限制酶切割作用 ( restriction enzyme digestion ) 29
2-8. DNA定序及序列比對分析 30
2-9. DNA片段純化作用 ( gel purification ) 30
2-10. DNA接合反應 ( ligation ) 31
2-11. 大腸桿菌中質體DNA的製備 32
2-12. DNA酒精沉澱作用 32
2-13. 細胞株及其培養 32
2-14. DNA轉染作用 ( transfection ) 36
2-15. 細胞萃取液之製備 37
2-16. 蛋白質定量 38
2-17. 膠凝體電泳 ( sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE ) 38
2-18. 西方墨點法 ( western blotting ) 39
2-19. 免疫共沉澱法 ( co-Immunoprecipitation，Co-IP ) 41
2-20. 免疫螢光染色 （immunofluorescence staining） 及雷射掃描共軛焦顯微鏡 ( laser confocal microscope ) 42
2-21. 細胞存活能力分析 ( MTT assay ) 43
2-22. 細胞爬行能力分析 ( migration assay ) 43
2-23. 細胞不貼附生長Anchorage-Independent Growth ( soft agar colony formation assay ) 44
2-24. 建立病毒 45
2-25. 細胞感染 ( infection ) 45
2-26. 抗生素篩選感染細胞株 46
2-27. 統計 46
肆、研究結果 47
一、在293T 細胞株中過度表現BAG2植體 47
二、以免疫共沉澱法 ( Co-immunoprecipitation，Co-IP ) 確認 Tid1和 BAG2之結合 47
三、以雷射掃描共軛焦顯微鏡 ( Laser confocal microscope ) 確認Tid1和BAG2是否在粒線體 ( sub-localization ) 48
四、分析口腔癌細胞株BAG2表現量 48
五、在SAS 細胞株中過度表現BAG2 49
六、探討在SAS 細胞株中過度表現BAG2之口腔癌細胞的存活率 ( viability ) 50
七、探討在SAS 細胞株中過度表現BAG2之口腔癌細胞的爬行能力 ( migration ) 50
八、利用Soft Agar探討BAG2過度表現的SAS癌細胞不貼附生長 ( anchorage-independent growth ) 之能力 51
九、在 OECM-1細胞株中過度表現 BAG2 51
十、探討在 OECM-1細胞株中過度表現BAG2之口腔癌細胞的存活率 ( viability ) 52
十一、利用Soft Agar探討BAG2過度表現的OECM-1癌細胞不貼附生長 ( anchorage - independent growth ) 之能力 52
十二、確認抑制BAG2基因表達質體的抑制效果 53
十三、探討在SAS 和OECM-1細胞株中shBAG2之口腔癌細胞的存活率 ( viability ) 53
十四、探討在SAS和OECM-1細胞中shBAG2之口腔癌細胞的增生能力 ( proliferation ) 54
十五、探討在SAS和OECM-1細胞株中抑制 BAG2之口腔癌細胞的爬行能力 ( migration ) 54
十六、利用 Soft Agar 探討在 SAS 和 OECM-1細胞株中抑制 BAG2的口腔癌細胞不貼附生長 ( anchorage - independent growth ) 之能力 54
伍、研究討論 55
一、利用免疫共沉澱 ( Co-immunoprecipitation ) 和雷射共軛焦顯微鏡 ( confocal microscopy ) 釐清 BAG2與 Tid1的關係 55
二、BAG2在口腔癌細胞中扮演的角色 56
三、BAG2在口腔癌細胞中是否會誘導細胞凋亡 ( apoptosis ) 57
四、BAG2與細胞自嗜 ( autophagy ) 的關係 57
五、使用臨床藥物測定BAG2在口腔癌細胞中的角色 58
六、未來目標 58
陸、參考文獻 59
柒、實驗圖表 66
捌、附錄 101

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