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研究生:李凱君
研究生(外文):Kai-chun Li
論文名稱:阻斷細胞周圍FibronectinMatrix與DPPIV間的結合作為具有潛力的抗轉移策略
論文名稱(外文):Blocking the Interaction between Pericellular Fibronectin Matrix and DPP IV as a Potential Anti-metastatic Strategy
指導教授:鄭宏祺鄭宏祺引用關係
指導教授(外文):Hung-chi Cheng
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:113
中文關鍵詞:第四型雙胜肽水解酶轉移纖連蛋白
外文關鍵詞:FNmetastasisDPP IV
相關次數:
  • 被引用被引用:1
  • 點閱點閱:252
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要--------------------------------------------------I
英文摘要-------------------------------------------------II
誌謝-----------------------------------------------------IV
目錄------------------------------------------------------V
表目錄---------------------------------------------------IX
圖目錄----------------------------------------------------X
附圖目錄------------------------------------------------XII
縮寫---------------------------------------------------XIII
第一章 緒論-----------------------------------------------1
1-1癌症轉移-----------------------------------------------1
1-2臟器特異性癌症轉移-------------------------------------2
1-3 FN/DPP IV與臟器特異性癌症轉移-------------------------3
1-4第四型雙胜肽水解酶
(Dipeptidyl peptidase IV; DPP IV; CD26)-------------------4
1-5纖連蛋白 (Fibronectin; FN)-----------------------------5
1-6核糖核酸干擾 (RNA interference; RNAi)------------------6
1-7 核糖核酸干擾機制--------------------------------------7
1-8核糖核酸之運送-----------------------------------------8
1-9腺病毒 (Adenovirus)------------------------------------9
1-10重組腺病毒載體---------------------------------------10
1-11研究動機---------------------------------------------10
第二章 材料與方法----------------------------------------12
2-1. 實驗材料--------------------------------------------12
2-1-1. 實驗動物------------------------------------------12
2-1-2. 細胞株--------------------------------------------12
2-1-3. 抗體----------------------------------------------12
2-1-4 . 培養液-------------------------------------------12
2-1-5. 緩衝液--------------------------------------------13
2-2. 實驗方法--------------------------------------------18
2-2-1. 繼代細胞------------------------------------------18
2-2-2. 冷凍保存細胞--------------------------------------19
2-2-3. 解凍細胞------------------------------------------19
2-2-4. 抽取細菌質體DNA-----------------------------------20
2-2-5. 電泳法回收DNA-------------------------------------21
2-2-6. 大腸桿菌勝任細胞製作 (competent cell preparation)-22
2-2-7. 限制.切割-----------------------------------------22
2-2-8. 接合作用 (Ligation) ------------------------------23
2-2-9. 菌株轉型作用 (Transform) -------------------------24
2-2-10. CaCl2細胞轉染------------------------------------24
2-2-11. 生產腺病毒---------------------------------------25
2-2-12. 腺病毒感染目標細胞4T1----------------------------26
2-2-13. 腺病毒MOI計算------------------------------------27
2-2-14. RNA萃取------------------------------------------27
2-2-15. 反轉錄聚合.連鎖反應 (RT-PCR) --------------------28
2-2-16. 黏著細胞Condition medium收集---------------------30
2-2-17. TCA濃縮蛋白--------------------------------------30
2-2-18. 黏著細胞蛋白質萃取-------------------------------31
2-2-19. 蛋白質定量---------------------------------------31
2-2-20. SDS-PAGE聚丙醯胺膠體蛋白質電泳法-----------------32
2-2-21. 西方墨點法---------------------------------------33
2-2-22. 黏著細胞表面免疫螢光染色-------------------------34
2-2-23. 懸浮細胞免疫螢光染色-----------------------------35
2-2-24. Microporation細胞轉染----------------------------36
2-2-25. 細胞黏著試驗 (Cell-cell adhesion) ---------------37
2-2-26. 表現MBP重組蛋白----------------------------------37
2-2-27. 純化MBP重組蛋白----------------------------------38
2-2-28. 生物素標記蛋白(Biotinylation) -------------------39
2-2-29. Far western assay (Gel overlay assay) -----------40
2-2-30. 親和性沉澱試驗 (Pull down assay) ----------------41
2-2-31. DP4A binding assay-------------------------------42
2-2-32. DP4A binding assay (FN fragments inhibit) -------43
第三章 實驗結果------------------------------------------44
3-1. 構築可辨認endo-FN序列之shRNA的scramble control------44
3-2. 獲得帶有FN shRNA及scramble shRNA之重組腺病毒--------44
3-3. 以帶有FN shRNA及scramble shRNA之腺病毒感染標的細胞
4T1------------------------------------------------------45
3-4. FN shRNA可抑制4T1細胞endo-FN合成--------------------45
3-5. Endo-FN確實參與癌細胞表面FN matrix組裝--------------46
3-6. 降低癌細胞表面FN matrix組裝可抑制癌細胞與DPP IV黏著-48
3-7. 降低癌細胞表面FN matrix可抑制癌細胞之肺臟轉移-------48
3-8. FN N端30及45kDa片段中數個重複片段存在DPP IV結合位點-50
3-9. DPP IV與Heparin在FN N端結合位點相似性很高且兩者可能
利用相同方式與FN N端重複片段進行結合---------------------50
3-10. 經由計算解離常數 (Kd)得知FN45-4與DPP IV及Heparin
之結合均具特異性且屬於typical ligand-receptor間的結合----51
3-11. FN N端DPP IV結合片段可抑制DP4A與細胞表面FN matrix
結合-----------------------------------------------------52
3-12. 結合FN C端及N端DPP IV結合片段可更加抑制DP4A與細胞表面
FN matrix結合--------------------------------------------52
3-13. 結論-----------------------------------------------53
第四章 討論----------------------------------------------55
4-1. FN在癌症轉移所扮演之角色----------------------------55
4-2. FN shRNA knock down癌細胞endo-FN對抑制細胞表面FN
matrix組裝之效率-----------------------------------------55
4-3. Endo-FN於FN matrix組裝中扮演的角色------------------57
4-4. 使用重組腺病毒載體之優缺點--------------------------57
4-5. FN之DPP IV結合位點----------------------------------59
4-6. 以Far western及pull down assay尋找FN N端重複片段之
DPP IV結合位點-------------------------------------------59
4-7. FN上之肝素 (heparin)與DPP IV的結合位點--------------62
第五章 參考文獻------------------------------------------64
第六章 表------------------------------------------------71
第七章 圖------------------------------------------------75
第八章 附圖---------------------------------------------104
第九章 自述---------------------------------------------113
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